Methods of using microfluidic positional encoding devices

Abstract

The invention relates to methods and compositions useful for routing and tracking multiple mobile units within a microfluidic device. Mobile units may be routed through a plurality of chemical environments, and the mobile units may be tracked to determine the path and / or environments that the mobile units have routed through. Mobile units may be routed in accordance with a predetermined algorithm. Mobile units may be routed through microfluidic devices in ordered flow. Mobile units routed through the microfluidic device can be used to perform various chemical reactions uniquely associated to the units, including without limitation peptide synthesis, enzymatic gene synthesis and gene assembly.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is the National Stage of International Application No. PCT / US2020 / 012627, filed Jan. 7, 2020, which claims the benefit of and priority to U.S. Provisional Application Nos. 62 / 789,505, filed Jan. 7, 2019, 62 / 789,506 filed on Jan. 7, 2019, 62 / 810,196 filed on Feb. 25, 2019, 62 / 811,506 filed on Feb. 27, 2019, and 62 / 863,712 filed on Jun. 19, 2019, all of which are incorporated by reference in their entirety.BACKGROUND OF THE INVENTION

[0002] In biology, chemistry, and other areas it is often desirable to both create large collections of chemical compounds or products and to evaluate the characteristics, properties, performance, or utility of these products. Historically, individual products were manufactured and characterized in separate vessels. Batch type procedures have been developed and disclosed that enable production of multiple products at a time. However, due to the cost, space requirements, and physical manipulations required, there has been a longstanding desire to develop alternative methods that can produce or evaluate very large libraries of products. Approaches such as split synthesis require encoding, randomness, redundancy, and underrepresentation problems in libraries. It can be time consuming, costly, or laborious to discover the identity of the product of interest associated with a unit. Further, encoding approaches have challenges relating to cost effectiveness, scalability, speed, and accuracy.

[0003] Since the advent of PCR in 1983, the uses of customized synthetic DNA have exploded to include a rich array of applications that impact the health of millions. Among these are noninvasive prenatal tests for fetal trisomies, tests for carriers of inherited diseases, and tests that allow the selection of the cancer treatments. Synthetic DNA is also a critical input into the development of therapeutic RNAs, aptamers, and antibodies, as well as gene editing. However, orders of large numbers of DNA fragments from existing DNA suppliers result in wait-times of days and sometimes many weeks. This latency results in enormous hidden financial and opportunity costs, and fundamentally slows the pace of development of new diagnostics and therapeutics.

[0004] Therefore, there is a need for a novel microfluidic technology to enable the rapid, massively parallel, cost-efficient synthesis of nucleic acids that is able to produce thousands of high-quality nucleic acid fragments in hours. Giving scientists access to one to thousands of nucleic acid fragments, nearly on demand, stands to dramatically accelerate the pace development of new diagnostics and therapeutics, lower cost by increasing overall productivity, and open up new research paradigms based on rapid design iteration that were not previously feasible.SUMMARY OF THE INVENTION

[0005] Disclosed herein are methods and compositions relating to tracking of mobile units within a microfluidic device. In various embodiments, the tracking of mobile units is achieved by controlling or recording the position, e.g. the relative position, of the mobile units, for example as the mobile units are moving through various compartments of the microfluidic device. The tracked mobile units may be split into the channels of a microfluidic device, for example by employing a router, such as a distributor, and recombined. The order of the mobile units upon recombination may be indicative of the path each mobile unit took through the microfluidic device. Individual channels of the microfluidic device may be used to perform reactions, such as synthesis reactions. Such reactions may be performed in parallel. Reagents for each reaction may be delivered to the individual channels, for example via separate reagent delivery channels. Suitable reaction conditions, such as temperature, pressure, and flow rate may be set in the individual channels.

[0006] In a first aspect, the methods and compositions described herein relate to tracking of mobile units within a microfluidic device. The tracking may comprise moving k mobile units through a first channel of a microfluidic device in a first order; splitting the k mobile units into z branch channels; and moving the k mobile units into a second channel in a second order.

[0007] Each of the k mobile units may be mappable to one of the z branch channels based on the second order. The k mobile units may further be moved from the second channel to the first channel. The second channel may be in fluidic communication with the first channel. The steps of moving k mobile units through a first channel of a microfluidic device in a first order, splitting the k mobile units into z branch channels, and moving the k mobile units into a second channel in a second order may be repeated n times. In some embodiments, n is or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. In some embodiments, n is 2. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. The mobile units may be beads, droplets, cells, bubbles, slugs or immiscible volumes. The beads may comprise glass or silica beads, metal beads, hydrogel or polymer beads, or chemically resistant polymer beads. The microfluidic device may comprise at least i channels having a largest cross-section no greater than x times the mean cross-section of the mobile units. In some embodiments, x is or is less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.02, 1.01, or 1. In some embodiments, i is or is greater than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, or 10000. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 500, 400, 300, 250, 200, 150, 100, 90, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 micrometers. In some embodiments, j is or is greater than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, or 10000. In some embodiments, the cross-section coefficient of variation for the k mobile units is or is less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%5, 5% 4%, 3%, 2%, 1%, or less. In some embodiments, a different set of reagents is delivered to each of a subset or all of the z branch channels. The one or more sets of reagents may comprise a 2′-deoxynucleoside phosphoramidite. The first order or the second order may be predetermined. In some embodiments, z is or is more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. Each of a subset or all of the z branch channels may comprise valves at one or both ends. One or more reagent channels may be configured to deliver reagents to each of a subset or all of the z branch channels. The delivery of reagents from at least one of the one or more reagent channels may be controlled by a valve. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, or 1000000. In some embodiments, k is or is less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. In some embodiments, k is between 2 and 500.

[0008] In a second aspect, the methods and compositions described herein relate to a microfluidic device and uses thereof. The microfluidic device may comprise a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels may be configured to accept mobile units from the first channel in a first order; and a second channel in fluidic communication with the set of z branch channels, wherein the second channel may be configured to accept mobile units from the set of z branch channels in a second order. The first or the second order may be controllable. The second order may be determinative of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The microfluidic device may comprise k mobile units. The microfluidic device may comprise a router, e.g. a distributor, between the first channel and the set of z branch channels. In some embodiments, z is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. In some embodiments, k is or is less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. In some embodiments, k is between 2 and 500.

[0009] In a third aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein the synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device. The microfluidic device may further comprise i fiducial marks. The configuration of the k mobile units may depend on the relative position of j mobile units with respect to the i fiducial marks. In some embodiments, i is or is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, j is or is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more.

[0010] A fourth aspect of the methods and compositions described herein relates to a system comprising computer comprising a computer-readable medium; and a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein the synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device; wherein the computer is configured to record data associated with the position of the k mobile units in the computer-readable medium repeatedly.

[0011] A fifth aspect of the methods and compositions described herein relates to a system comprising a computer comprising a computer-readable medium and a microfluidic device. The microfluidic device may comprise a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; and a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order. The second order may be determinative or predictive of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The computer may be configured to record data associated with the position of the mobile units in the computer-readable medium repeatedly.

[0012] In a sixth aspect, the methods and compositions described herein relate to routing of mobile units within a microfluidic device. The method may comprise a) routing k mobile units through a first channel of a microfluidic device in a first order; b) distributing the k mobile units into z branch channels; and c) routing the k mobile units into a second channel in a second order. The routing in step a may be performed in accordance with a predetermined unit routing algorithm through the microfluidic device for at least a subset of the k mobile units. The unit routing algorithm may comprise a routing selection at at least one branch point of the microfluidic device. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels based on unit tracking information from at least one detector configured to track the movement of mobile units inside the microfluidic device. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels based on the second order. At least a subset of the k mobile units in step c may comprise all of the k mobile units. The first channel and the second channel may be the same. Between steps b and c, the flow direction of at least a subset of the k mobile units may be reversed. In step b, at least one unit may be routed into a first branch channel through a first branch channel end and in step c, the at least one unit may be routed out of the first branch channel through the first branch channel end. In step b, at least one unit may be routed into a first branch channel through a first branch channel end and, in step c, the at least one unit may be routed out of the first branch channel through a second branch channel end that is different than the first branch channel end. The method may further comprise routing the k mobile units from the second channel to the first channel. The second channel may be in fluidic communication with the first channel. The method may further comprise repeating steps a-c n times. n may be 2. n may be 2 to 10. n may be 10 to 100. n may be 100 to 1000. n may be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. Units may be beads. The mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. The beads comprise glass beads or polymer beads. The microfluidic device may comprise i channels having a largest cross-section x times the mean cross-section of the k mobile units. i may be 2-10000. x may be 1.05-2.0. i may be 2-100. i may be 100-1000. The microfluidic device may comprise at least i channels having a largest cross-section no greater than x times the mean cross-section of the k mobile units. The mobile units may be beads. x may be or may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or less. X may be or may be more than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or more. i may be or may be more than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, 10000, or more. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 200 micrometers. j may be 2 to 10000. The largest cross-section of the at least j channels may be no greater than 10 micrometers. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 200 micrometers. j may be 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 5000, 10000, or more. The cross-section coefficient of variation for the k mobile units may be 1% to 20%. The cross-section coefficient of variation for the k mobile units may be 2% to 5%. The cross-section coefficient of variation for the k mobile units may be less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The method may further comprise delivering different reagents to each of the z branch channels. The reagents may comprise a 2′-deoxynucleoside phosphoramidite. The method may further comprise directing at least one mobile units into a side channel. The method may further comprising directing the at least one mobile units in the side channel to the second channel. The first order may be predetermined. The second order may be predetermined. z may be 2-10 z may be 10-100. z may be 100-1000. z may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. z may be less than 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. Each of the z branch channels may be capped by valves or unit stops on one or two ends. One or more reagent channels may be configured to deliver reagents to each of the z branch channels. Delivery of reagents from at least one of the one or more reagent channels may be controlled by a valve. Delivery of reagents from at least one of the one or more reagent channels may be controlled by application of differential pressures to selected points in the z branch channels and the reagent channels. k may be between 2 and 1000000. k may be 2-5000000. k may be 20-100. k may be 100-1000. k may be 10000-100000. k may be 100000-1000000. k may be between 2 and 500. K may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, or 20. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the second order may be verified using the at least one unit's label. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the first order may be verified using the at least one unit's label. The at least one mobile unit may comprise at least two mobile units. The labels of the at least two mobile units may be not unique.

[0013] In a seventh aspect, the methods and compositions described herein relate to a microfluidic device comprising: a) a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; and b) a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order; wherein the second order is determinative of the particular branch channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The first order or the second order may be controllable. The device may further comprise k mobile units. The device may further comprise a distributor between the first channel and the set of z branch channels. z may be between 2 and 50. z may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. z may be less than 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. k may be between 2 and 500. k may be between 2 and 5000000. k may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, 5000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less.

[0014] In an eight aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device.

[0015] In a ninth aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a treatment history for each of the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device. The treatment history may comprise a light treatment history, a heat treatment history, an enzymatic treatment history, a cleavage treatment history, an isomerization history, an acetylation history, a synthesis history, an amplification history, or a reaction history. The microfluidic device may further comprise i fiducial marks. The configuration of the k mobile units may depend on the relative position of j mobile units with respect to the i fiducial marks. i may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. i may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. j may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. j may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or less.

[0016] In a tenth aspect, the methods and compositions described herein relate to a system comprising a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a treatment history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device; wherein the computer is configured to record data associated with the position of the k mobile units in the computer-readable medium repeatedly. The treatment history may comprise a light treatment history, a heat treatment history, an enzymatic treatment history, a cleavage treatment history, an isomerization history, an acetylation history, a synthesis history, an amplification history, or a reaction history.

[0017] In an eleventh aspect, the methods and compositions described herein relate to a system comprising: a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising i) a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; ii) a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order; wherein the second order is determinative of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order; and wherein the computer is configured to record data associated with the position of the mobile units in the computer-readable medium repeatedly.

[0018] In a twelfth aspect, the methods and compositions described herein relate to a method of tracking, the method comprising: a) moving k mobile units through a first channel of a microfluidic device in a first order; b) routing at least a subset of the k mobile units within the microfluidic device, thereby creating a second order; c) performing a comparison of the second order to a predesignated post-routing order; and d) separating j mobile units into a correction area based on the comparison of step c by separating the j mobile units from a remainder of the at least a subset of the k mobile units; wherein each of the remainder of the at least a subset of the k mobile units is mappable to a routing path. The routing path may comprise the location of a mapped mobile unit after the routing step in step b. The routing path may comprise the location of a mapped mobile unit before the routing step in step b. The location of a mobile unit may comprise the unit's relative positional order with respect to m mapping mobile units. M may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more. m may be less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The m mapping mobile units may comprise the m closest mobile units to the mapped mobile unit along a fluidically connected path originating from the mapped mobile unit. Routing may comprise distributing into at least one branch channel of the microfluidic device. Routing may comprise merging from a plurality of branch channels of the microfluidic device. The correction area may comprise a channel of the microfluidic device. The method may further comprise merging at least one of the j mobile units with at least a subset of the remainder of the at least a subset of the k mobile units. k may be between 2 and 500. k may be between 2 and 100000. k may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the second order may be verified using the at least one unit's label. At least one mobile unit of the k mobile units may comprise a label. The position of the at least one mobile unit in the first order may be verified using the at least one unit's label. The at least one mobile unit may comprise at least two mobile units. The labels of the at least two mobile units may be not unique. j may be between 1 and 1000000. j may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000, or more. j may be less than 1000000, 100000, 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 7, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The method may further comprise repeating steps a-c n times. n may be 2. n may be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be less than 100, 750, 500, 400, 300, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. Beads may comprise glass beads or polymer beads. The comparison in step c may comprise verifying by at least one detector the position of at least one unit in the first order. The comparison in step c may comprise verifying by at least one detector the position of at least one unit in the second order. The comparison in step c may comprise counting units by at least one detector after the routing in step b is performed on one or more units, thereby generating a list of unit counts, and comparing the list of unit counts to an expected list of unit counts based on the predesignated post-routing order. The comparison in step c may comprise detecting one or more labels on one or more units by at least one detector after the routing in step b is performed on one or more units, thereby generating a list of detected unit labels, and comparing the list of detected unit labels to an expected list of unit labels based on the predesignated post-routing order.

[0019] In a thirteenth aspect, the methods and compositions described herein relate to a system comprising a) a microfluidic channel configured to carry beads in a carrier fluid; b) a detector configured to detect signals from a detection path through the microfluidic channel; and c) computer operably connected to the detector; wherein the system is calibrated to identify the signal of an isolated single bead in the microfluidic channel passing through the detection path. The system may be further calibrated to identify the signal of n adjacent beads in the microfluidic channel passing through the detection path. n may be 2 to 100. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. n may be less than 100, 90 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The system may be further calibrated to identify the signal of a gas bubble or a dust particle in the microfluidic channel passing through the detection path. The system may further comprise a router configured to route one or more beads from the microfluidic channel. The system may be configured to send a desired routing signal to the router to effectuate routing upon identification of an isolated single bead, a plurality of adjacent beads, a gas bubble or dust particle passing through the detection path. The router may comprise a distributor. The system may further comprise a bead spacer. The bead spacer may be configured to space beads flowing adjacently within the microfluidic channel. The system may further comprise a second microfluidic channel. The router may be configured to route beads into the second microfluidic channel. The router may comprise a merger.

[0020] In a fourteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising: a) a primary channel; b) a branch point; c) a first branch channel, wherein the first branch channel is fluidically connected to the primary channel through the branch point; and d) a first router configured to route units flowing in the primary channel into the first branch channel. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel and a location within the first branch channel. The device may further comprise a second branch channel, wherein the second branch channel is fluidically connected to the primary channel through the branch point. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel, a location within the first branch channel, and a location within the second branch channel. The first router may be configured to route units from the primary channel into the second branch channel by causing a pressure differential between one or more locations within the primary channel, a location within the first branch channel, and a location within the second branch channel. The device may further comprise z branch channels. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel and a location within the first branch channel, and pressure differentials between one or more locations within the primary channel and a location within each of the z branch channels. The router may comprise a network of fluidic outlets configured to connect to pressure controllers, such that the router is capable to regulate the fluidic pressure within channels that are connected through the branch point. The branch channels may connect to the primary channel at separate positions of the primary channel. The device may further comprise a second router configured to route units from at least one of the branch channels to the primary channel. The first router may comprise the second router. The second router may comprise a merger.

[0021] In a fifteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising a microfluidic channel holding k mobile units wherein the microfluidic device is configured to maintain the relative positional order of the k mobile units and wherein the microfluidic channel is configured to flow the k mobile units in a carrier fluid. There may be a distance greater than a minimum distance between each pair of the k mobile units measured along a path of fluidic connection. The minimum distance may be at least 1.5 times the mean diameter of the pair of the k mobile units. The minimum distance may be 2 to 10000 times the mean diameter of the pair of the k mobile units. The minimum distance may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 100, 1000, 5000, 10000, or more times the mean diameter of the pair of the k mobile units. The minimum distance may be less than 10000, 5000, 1000, 100, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, times the mean diameter of the pair of the k mobile units, or less. The width of the microfluidic channel may be at least 2 times the average diameter of the k mobile units. The width of the microfluidic channel may be at least 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000 times the average diameter of the k mobile units or more. The width of the microfluidic channel may be less than 50000, 10000, 1000, 100, 90, 80, 70, 60, 50, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2 times the average diameter of the k mobile units or less.

[0022] In a sixteenth aspect, the methods and compositions described herein relate to a method of separating beads in a microfluidic device, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second channel, wherein the first microfluidic channel and the second channel are connected by a bead spacer; b) moving a plurality of beads through the first microfluidic channel toward the bead spacer; c) passing a first bead and a second bead serially through the bead spacer into the second channel; and d) moving a carrier fluid through the second channel such that a desired length of carrier fluid is spaced between the first bead and the second bead in the second channel. Steps a-d may be repeated at least n times. n may comprise 2 to 1000000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more. n may be at most 10000000, 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The plurality of beads may comprise 2 to 1000000 beads. The plurality of beads may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more beads. The plurality of beads may comprise at most 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 21, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer beads. The desired length of carrier fluid may be 1 to 1000 times the average size of the plurality of beads. The desired length of carrier fluid may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the average size of the plurality of beads, or greater. The desired length of carrier fluid may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the average size of the plurality of beads, or less. The plurality of beads may comprise 2 to 1000000 beads. The plurality of beads may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more beads. The plurality of beads may comprise at most 10000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer beads. The first channel width may be 1 to 2 times the average diameter of the beads. The first channel width may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or 1.01 times the average diameter of the beads, or less. The first channel width may be more than 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the beads, or more. The second channel width may be 1.01 and 100 times the average diameter of the beads. The second channel width may be at least 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average diameter of the beads, or greater. The second channel width may be at most 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or 1.01 times the average diameter of the beads, or smaller. The carrier fluid speed may be less than 50 meters / sec, 10 meters / sec, 1 meters / sec, 100 millimeters / sec, 10 millimeters / sec, 11 millimeters / sec, 0.1 millimeters / sec, or 0.01 millimeters / sec, or less. The carrier fluid speed may be at least 0.01, 0.1, 1, 10, 100 millimeters / sec, 1, 10, or 50 meters / sec, or more. The first and the second bead may be passed through the bead spacer within less than 10 sec, 1 sec, 0.1 sec, 0.01 sec, 1 msec, 0.1 msec, or 0.01 msec, or faster.

[0023] In a seventeenth aspect, the methods and compositions described herein relate to a microfluidic device comprising a microfluidic channel holding k mobile units wherein the microfluidic device is configured to maintain the relative positional order of the k mobile units and wherein the microfluidic channel is configured to flow the k mobile units in a carrier fluid. The width of the microfluidic channel may be 0.05 to 2 times the average diameter of the k mobile units measured outside of the microfluidic channel. The width of the microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.01, 1, 0.95, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.4, 0.3, 0.2, 0.1, or 0.05 times the average diameter of the k mobile units measured outside of the microfluidic channel, or smaller. The width of the microfluidic channel is more than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1, 1.01, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 1.95 times the average diameter of the k mobile units measured outside of the microfluidic channel, or greater. The device may be configured to move the k mobile units within the microfluidic channel along a moving direction of the microfluidic channel. There may be a center to center distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel of less than 2 times the average diameter of the k mobile units. The center to center distance may be 0.01 to 1.9 times the average diameter of the k mobile units. The center to center distance may be less than 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65. 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times the average diameter of the k mobile units, or less. The center to center distance may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the average diameter of the k mobile units, or greater. The device may be configured to move the k mobile units within the microfluidic channel along a moving direction of the microfluidic channel. The shortest distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65. 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The shortest distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The maximum deviation from the average width of the microfluidic channel may be less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.10%, or less. The maximum deviation from the average width of the microfluidic channel may be more than 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more. The coefficient of variance in the diameter of the k mobile units may be less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The coefficient of variance in the diameter of the k mobile units is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0024] In an eighteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein the coefficient of variance in the diameter of the k mobile units is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The coefficient of variance in the diameter of the k mobile units may be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more.

[0025] In a nineteenth aspect, the methods and compositions described herein relate to a method of sorting, the method comprising: a) providing k mobile units; b) introducing the k mobile units into a unit size sorter; c) separating a subset of k mobile units having sizes falling outside of a predetermined range of unit size from the remainder of the k mobile units; and d) introducing at least a subset of the remainder of the k mobile units into a microfluidic device. The upper limit of the predetermined range of unit size may be less than 1.3, 1.25, 1.2, 1.15, 1.14, 1.13, 1.12, 1.11, 1.1, 1.09, 1.08, 1.07, 1.06, 1.05, 1.03, or 1.02 times the lower limit of the predetermined range, or less. The upper limit of the predetermined range of unit size may be more than 1.02, 1.03, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.2, 1.25, or 1.3 times the lower limit of the predetermined range, or more.

[0026] In a twentieth aspect, the methods and compositions described herein relate to a method of separating units in a microfluidic device, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second channel, wherein the first microfluidic channel and the second channel are connected by a unit spacer; b) moving a plurality of units through the first microfluidic channel toward the unit spacer; c) passing a first unit and a second unit serially through the unit spacer into the second channel; and d) moving a carrier fluid through the second channel such that a desired length of carrier fluid is spaced between the first unit and the second unit in the second channel. The steps a-d may be repeated at least n times. n may be 2 to 1000000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more. n may be at most 10000000, 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The plurality of units may comprise 2 to 1000000 units. The plurality of units may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000 or more units. The plurality of units may comprise at most 1000000, 100000, 100000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer units. The desired length of carrier fluid may be 1 to 1000 times the average size of the plurality of units. The desired length of carrier fluid may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the average size of the plurality of units, or greater. The desired length of carrier fluid may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the average size of the plurality of units, or smaller. The first channel width may be 1.1 to 2 times the average diameter of the units. The first channel width may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 times the average diameter of the units, or smaller. The first channel width may be more than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the units, or greater. The second channel width may be 1.05 to 100 times the average diameter of the units. The second channel width may be at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average diameter of the units, or greater. The second channel width may be at most 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.05 times the average diameter of the units, or smaller. The carrier fluid speed may be at least 0.01, 0.1, 1, 10, 100 millimeters / sec, 1, 10, or 50 meters / sec, or faster. The carrier fluid speed may be less than 50 meters / sec, 10 meters / sec, 1 meters / sec, 100 millimeters / sec, 10 millimeters / sec, 11 millimeters / sec, 0.1 millimeters / sec, or 0.01 millimeters / sec, or slower. The first and the second unit may be passed through the unit spacer within 0.01 msec to 10 sec. The first and the second unit may be passed through the unit spacer within less than 10 sec, 1 sec, 0.1 sec, 0.01 sec, 1 msec, 0.1 msec, 0.01 msec, or faster. The microfluidic device may be configured to maintain the relative positional order of the plurality of units. The plurality of units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. Beads may comprise glass beads or polymer beads.

[0027] In a twenty first aspect, the methods and compositions described herein relate to a system comprising: a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising r routers and c microfluidic channels in fluidic connectivity, wherein the r routers are configured to route k mobile units through at least a subset of the c microfluidic channels; and c) d detectors operably connected to the computer, wherein the detectors are configured to detect signals from detection paths through the at least c microfluidic channels or the at least r routers; wherein the computer is configured to record data associated with detected signals from the at least d detectors in the computer-readable medium repeatedly and to generate routing paths for at least a subset of the k mobile units. c may be 2 to 1000. c may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. c may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less. d may be 2 to 1000. d may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. d may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. r may be 2 to 1000. r may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. r may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. k may be 2 to 1000000. k may be at least at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The system may be further configured to route at least j units of the k mobile units to a first channel of the c microfluidic channels n times. n may be 2 to 1000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. n may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. j may be 2 to 5000000. j may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, 5000000, or more. j may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The k mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. The c routers may comprise one or more distributors, mergers, or spacers. The routing path may comprise the location of a mapped mobile unit downstream of a router. The routing path may comprise the location of a mapped mobile unit upstream of a router. The location of a mobile unit may comprise the unit's relative positional order with respect to m mapping mobile units. m may be 1 to 100. m may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more. m may be at most 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The m mapping mobile units may comprise the m closest mobile units to the mapped mobile unit along a fluidically connected path originating from the mapped mobile unit. The r routers may be configured to route mobile units in accordance with a predetermined unit routing algorithm through the microfluidic device. The computer may be configured to perform a comparison between a first post-routing order for the at least a subset of the k mobile units after a routing event by at least one of the r routers and a predesignated post-routing order. The computer may be configured to generate routing paths for i of the at least a subset of the k mobile units based on the comparison. The r routers may be configured to route i mobile units in accordance with the routing paths for the i mobile units. i may be 2 to 1000000. i may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. i may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The r routers may be configured to separate j mobile units from a remainder of the at least a subset of the k mobile units into a correction area based on the comparison. The r routers may be configured to route mobile through the microfluidic device randomly.

[0028] In a twenty second aspect, the methods and compositions described herein relate to a method of tracking, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second microfluidic channel in fluidic connection with the first microfluidic channel; and b) routing k mobile units through the first microfluidic channel into the second microfluidic channel in ordered flow. The first microfluidic channel and the second microfluidic channel may be the same. The first microfluidic channel and the second microfluidic channel may be connected by a union, unit spacer, distributor, or merger. The microfluidic device may further comprise a third microfluidic channel. The method may further comprise routing the plurality of mobile units through the second microfluidic channel into the third microfluidic channel in ordered flow. The second microfluidic channel and the third microfluidic channel may be the same. The first microfluidic channel and the third microfluidic channel may be the same. The second microfluidic channel and the third microfluidic channel may be connected by a union, unit spacer, distributor, or merger. The width of the first microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the first microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the first microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the second microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the second microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the second microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller. The width of the third microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the third microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the third microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the first microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the first microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the first microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller. The width of the second microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the second microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the second microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the third microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the third microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the third microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller.

[0029] In a twenty third aspect, the methods and compositions described herein relate to a method of synthesizing oligomers associated with mobile units, the method comprising: a) routing k mobile units through a first channel of a microfluidic device in a first order; b) distributing at least a subset of the k mobile units into at least z branch channels; and c) routing the at least a subset of the k mobile units into a second channel in a second order; wherein at least a subset of the k mobile units are functionalized with a group suitable to synthesize an oligomer; wherein at least a subset of the k mobile units are mappable to a path comprising a specific one of the z branch channels; wherein at least a subset of the k mobile units are subjected to reaction conditions comprising conditions for a step of a synthesis reaction inside the z branch channels; and wherein steps a-c are repeated for n cycles. In some embodiments, the synthesis reaction comprises a nucleic acid synthesis reaction or a peptide synthesis reaction. In some embodiments, the reaction conditions comprise an enzyme. In some embodiments, the enzyme is selected from a terminal deoxynucleotidyl transferase, a thermostable DNA polymerase, a DNA polymerase theta, a Poly(A) polymerase, and a DNA polymerase encoded by a variant of the 9°N DNA Polymerase gene from Thermococcus species 9°N-7. In some embodiments, the variant of the 9°N DNA Polymerase gene comprises the 9°N (D141A / E143A / A485L) DNA Polymerase gene or the 9°N (E143D) DNA Polymerase gene. In some embodiments, the enzyme is conjugated to a nucleotide or a nucleotide analog. In some embodiments, the reaction conditions comprise a nucleotide or a nucleotide analog. In some embodiments, at least a subset of the k mobile units are functionalized with an initiator nucleic acid or a nascent oligonucleotide. In some embodiments, the nucleic acid synthesis reaction is a template independent nucleic acid synthesis reaction. In some embodiments, the method further comprises performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal nucleotide of initiator nucleic acids or nascent oligonucleotides associated with at least a subset of the k mobile units and a new nucleotide or nucleotide analog in the presence of a transferase enzyme. In some embodiments, the new nucleotide or nucleotide analog comprises a blocking moiety. In some embodiments, the method further comprises performing a deblocking reaction thereby removing the blocking moiety from the newly incorporated nucleotide or nucleotide analog. In some embodiments, the method further comprises one or more steps selected from the group consisting of a washing step, a modification step, a cleaving step, and a capping step. In some embodiments, two or more of the steps selected from the group consisting of the coupling reaction, the deblocking reaction, the washing step, the modification step, the cleaving step, and the capping step are performed in different cycles. In some embodiments, the oligomers are oligonucleotides and wherein the method further comprises assembling the oligonucleotides into genes. In some embodiments, assembling of the oligonucleotides into genes is performed using one or more method selected from the group consisting of polymerase-cycling assembly, enzymatic gene assembly, annealing and ligation reaction, shotgun ligation, shotgun ligation and co-ligation, gene synthesis via one strand, template directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron technology, Sloning building block technology, Golden Gate assembly, Dual-Asymmetric (DA) PCR, Overlap Extension (OE) Asymmetric PCR, Thermodynamically-Balanced Inside Out (TBIO), Two-Step (DA+OE), Polymerase Assembly Multiplexing (PAM), One-Step Simplified Gene Synthesis, Single Molecule PCR, TopDown Real-Time Gene Synthesis, Two-step Ligation and PCR, Brick-based assembly, Sequence- and Ligation-Independent Cloning (SLIC), Transformation-associated Recombination, Biobrick assembly, PCR-based two-step DNA synthesis (PTDS), successive PCR method. In some embodiments, the reaction conditions comprise one or more of reagents selected from the group consisting of an amino acid, a dipeptide, a polypeptide, and a carbodiimide. In some embodiments, the method further comprises performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal end of nascent peptides associated with at least a subset of the k mobile units and a new amino acid, dipeptide or polypeptide. In some embodiments, the method further comprises performing one or more step selected from the group consisting of a capping step, a washing step, and a deprotecting step. In some embodiments, two or more of the steps selected from the group consisting of the coupling reaction, capping step, washing step and the deprotecting step are performed in different cycles. In some embodiments, the same z branch channels are used in at least two of the n cycles. In some embodiments, n is 2. In some embodiments, n is 2 to 10. In some embodiments, n is 10 to 100. In some embodiments, n is 100 to 200. In some embodiments, n is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, or 1000. In some embodiments, n is at most 1000, 750, 500, 400, 300, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. n may fall within a range bounded by any of the foregoing values, e.g. 100-200, 200-300, 300-400, 400-500, 500-1000. In some embodiments, z is 2-100. In some embodiments, z is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. In some embodiments, z is at most 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2. z may fall within a range that is bounded by any of the foregoing values, e.g. 2-100, 2-16, 4-20, 2-24, etc. In some embodiments, k is at most 1000000000000, 100000000000, 10000000000, 1000000000, 100000000, 10000000, 1000000, 100000, 10000, 1000, 500, 100, or less. In some embodiments, k is between 20-500. In some embodiments, k is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may fall within a range that is bounded by any of the foregoing values, e.g. 2-50000, 10-50, 20-500 etc. In some embodiments, the mobile units are selected from the group consisting of beads, droplets, cells, bubbles, slugs, immiscible volumes, glass beads, polymer beads, cross-linked beads, cross-linked polymer beads, divinylbenzene cross-linked polymer beads, and divinylbenzene cross-linked polystyrene beads. In some embodiments, the first order is different in at least two of the n cycles. In some embodiments, the second order is different in at least two of the n cycles. In some embodiments, the first channel is the same as the second channel.

[0030] In a twenty fourth aspect of the methods and compositions described herein relate to a composition comprising n cross-linked beads wherein the coefficient of variation for bead diameter is less than 20%. In some embodiments, the coefficient of variation for bead diameter is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. In some embodiments, the mean diameter of the beads is between 0.1-500 μm. In some embodiments, the mean diameter of the beads is between 10-50 μm. In some embodiments, the mean diameter of the beads is between 20-150 μm. In some embodiments, the mean diameter of the beads is at least 0.1 μm, 1 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 150 μm 200 μm, 300 μm, 400 μm, 500 μm, or greater. In some embodiments, the mean diameter of the beads is at most 500 μm, 400 μm, 300 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 75 μm, 70 μm, 60 μm, 50 μm, 40 m, 30 μm, 20 μm, or smaller. The mean diameter of the beads may fall within a range bounded by any of the foregoing values, e.g. 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 20-50 μm, 80-90 μm, 90-100 μm, 0.1-400 μm, etc. In some embodiments, the beads are functionalized with a group suitable for oligomer synthesis. In some embodiments, the beads are functionalized with one or more groups selected from the group consisting of amine, hydroxyl, chloromethyl, aminomethyl, benzhydrodrylamino, silane, alkylsilane, and carboxyl groups. In some embodiments, the beads are cross-linked at a molar cross-linker ratio of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more. In some embodiments, the beads are cross-linked at a molar cross-linker ratio of at most 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or less. The beads may be cross-linked at a molar cross-linker ratio that falls within any range bounded by the foregoing values, e.g. 20-40%, 15-50%, 25-45% etc. In some embodiments, the beads are cross-linked using one or more cross-linkers selected from the group consisting of divinylbenzene, glutaraldehyde, formaldehyde, an epoxy compound, dialdehyde, and dichloroethane. In some embodiments, the beads are cross-linked using radiation or oxidation. In some embodiments, the variation in bead diameter suspended in organic solvent versus aqueous solvent is less than 80%, 30%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less. In some embodiments, the variation in bead diameter suspended in organic solvent versus aqueous solvent is greater than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 30%, 50%, or more. Variation in bead diameter suspended in organic solvent versus aqueous solvent may fall within a range bounded by any of the foregoing values, e.g. 0.5-2%, 1-5%, 2-10%, 1-50% etc. In some embodiments, the organic solvent is selected from the group consisting of toluene, acetonitrile, toluene, dichloromethane, tetrahydrofuran (THF), pyridine, N-methyl pyrrolidinone (NMP), 2,6-lutidine, carbon disulfide, 1,2-dichloroethane, 1,1-dichloroethane, chloroform, dimethylformamide, dimethylacetamide, dimethylsulfoxide (DMSO), ethylene carbonate, 1,4-dioxane, DME (1,2-dimethoxyethane), nitromethane, methyl tert-butyl ether, methyl ethyl ketone (butanone), and dichloromethane. In some embodiments, n is at least 500, 1000, 10000, 100000, 1000000, 5000000, 10000000, 100000000, 1000000000, 10000000000, 100000000000, 1000000000000 or more. In some embodiments, n is at most 1000000000000, 100000000000, 10000000000, 1000000000, 100000000, 10000000, 1000000, 100000, 10000, 1000, or less. n may fall within a range bounded by any of the foregoing values, e.g., 500-10000, 1000-10000000000, 10000-1000000000000 etc. In various embodiments, bead compositions described herein are contained in a microfluidic device.

[0031] In a twenty fifth aspect the methods and compositions described herein relate to a system comprising: a) a microfluidic device comprising i delivery channels each in fluidic communication with a different set of z branch channels, wherein each of the sets of z branch channels is configured to accept a plurality of mobile units in a first order from one of the i delivery channels through a branch point; b) one or more routers configured to route mobile units into one of the z branch points at the first branch point; and c) a controller configured to control the one or more routers to route mobile units into one of the z branch points at the first branch point; wherein the first order is determinative of the particular branch channel of the set of z branch channels into which the controller is configured to control the one or more routers to route a specific mobile unit. The system may further comprise j outlet channels in fluidic communication with some or all of the set of z branch channels, wherein the outlet channels are configured to accept mobile units from the branch channels in a second order. In some embodiments, the second order is determinative of the particular branch channel of the set of z branch channels that is configured to deliver a specific mobile unit into one of the outlet channels. In some embodiments, the delivery channels and the outlet channels are the same. In some embodiments, at least a subset of the one or more routers comprise one or more microfluidic structures configured to generate a vortex. In some embodiments, the microfluidic structures configured to generate a vortex are positioned a distance from the branch point that is at least 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 1000 m, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or greater. In some embodiments, the microfluidic structures configured to generate a vortex are positioned a distance from the branch point that is at most 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, or smaller. The microfluidic structures configured to generate a vortex may be positioned a distance from the branch point that falls within a range bounded by any of the foregoing values, e.g. 100 μm-2.3 mm, 200 μm-450 μm, 150 μm-750 μm, etc. In some embodiments, at least a subset of the one or more routers comprise a thermal bubble forming apparatus. In some embodiments, z is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. In some embodiments, z is at most 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2. z may fall within a range bounded by any of the foregoing values, e.g. 2-50, 4-20, 3-10, etc. In some embodiments, i is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000 or more. In some embodiments, i is at most 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. i may fall within a range bounded by any of the foregoing values, e.g. 2-30, 3-20, 10-500, etc In some embodiments, j is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000 or more. In some embodiments, j is at most 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. j may fall within a range bounded by any of the foregoing values, e.g. 1-10, 5-20, 10-100, etc In some embodiments, i equals j.

[0032] In a twenty sixth aspect, the methods and compositions described herein relate to a method of DNA assembly, comprising: disposing a solid phase support column in a chamber of a microfluidics circuit; performing an enzymatic reaction in a channel of the microfluidics circuit to produce a reaction mixture, the channel fluidly coupled to the chamber; and flowing the reaction mixture over the solid phase support column in the chamber to capture assembled oligos. In some embodiments, the method further comprises flowing a wash mixture over the solid phase support column to remove remaining portions of the reaction mixture. In some embodiments, the method further comprises flowing an elution fluid over the solid phase support column to elute the assembled plurality of oligonucleotides.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] These and other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures, wherein:

[0034] FIG. 1 provides an illustrative example of a microfluidic device comprising a first primary channel having a plurality of ordered mobile units, such as beads. A router, e.g. a distributor, (triangle) at the connection of the first channels with two branch channels can serve to direct each of the mobile units into one of the two branch channels. Valves in the two branch channels may be configured to control entry and exit of the mobile units. Reagents may be delivered to the two branch channels via reagent delivery channels. Delivery of reagents may be controlled with a valve. This configuration can be representative of one of many iterative steps a plurality of beads may undergo through the microfluidic device. The circles with numbers depict units with unit ID numbers; the rectangles depict valves; and the triangles depict routers, e.g. distributors.

[0035] FIG. 2 provides an illustrative example of a microfluidic device. Mobile units 1-6 from a first channel are being directed deterministically into one of two branch channels by using a router, e.g. a distributor. Beads 7-9 are arranged in the first channel, soon to enter the router. The router is programmed to deliver beads 7-9 the positions indicated by the hashed-circles. Once the mobile units are distributed into the branch channels, reagents, such as synthesis reagents may be circulated through the two branch channels that will be holding the mobile units.

[0036] FIG. 3 provides an illustrative example of a snapshot of tracked circulating of mobile units through split channels of a microfluidic device. The order of the mobile units in the channel as the mobile units are about to start a new round is different than the order shown in FIG. 1. The order of the mobile units as they are recirculated back to the first channel may be set in a deterministic manner. The position or relative position of specific mobile units may be known. In this illustrative example, the mobile units are being prepared to be distributed again into the branch channels that may be set to host a pre-assigned sequence of chemistries.

[0037] FIG. 4 provides an illustrative example of a microfluidic device wherein mobile units are split into four branch channels passing through two sets of successive routers, e.g. distributors. A device configuration with four branch channels may be used to synthesize nucleic acids in or on the mobile units by successive circulation of the mobile units through the branch channels. Dedicated reagent delivery channels may each provide one of four building blocks for nucleic acid synthesis.

[0038] FIG. 5 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. Valves in each of the four channels may control exit and entry of the mobile units and create a reaction chamber for a reaction cycle comprising chemical modification of the units when closed. Units released from one or more of the reagent chambers may be merged with the units released from another reaction chamber at successive branch points, resulting in combination of the units in the four channels into two channels.

[0039] FIG. 6 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. A detector in the two channels after the first router may interrogate the units as they pass through the channels. The data may be sent to a computer for storage and image processing.

[0040] FIG. 7 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. In this example, the units may be distributed into a reaction cluster comprising four reaction chambers with three consecutive valves: a first valve, a middle valve, and a last valve. These valves may form two reaction chambers in each channel, resulting in eight total reaction chambers in the reaction cluster.

[0041] FIG. 8 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. After the units undergo a reaction cycle in some or all of the reaction chambers, the units may be re-combined by flowing them through channels that merge, according to an algorithm or randomly. In this example, the two middle channels merge with each other first, before merging with the left (top) and the right (bottom) channels.

[0042] FIG. 9 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of routers, e.g. distributors. In this example, the units are distributed into different channels with varying numbers of reaction chambers.

[0043] FIG. 10 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. The reaction chambers may include additional features not shown, as indicated by the broken lines in the channel.

[0044] FIG. 11 provides an illustrative example of a microfluidic device with two consecutive reaction clusters.

[0045] FIG. 12 provides an illustrative example of a microfluidic device with two consecutive reaction clusters. In this example, the reaction chambers may include additional features not shown, as indicated by the broken lines in the channel.

[0046] FIG. 13 provides an illustrative example of a microfluidic device with a plurality of reaction zones. Units distributed into the different reaction zones may undergo the same reaction, different reactions, or no reaction. Reactions may occur simultaneously, consecutively, or at different times.

[0047] FIG. 14 provides an illustrative example of a microfluidic device with unit spacers wherein mobile units are distributed into two branch channels. The unit spacer(s), unit stop(s), and / or the pressure controller(s) and / or regulator(s) may be used to space and distribute units into branch channels and merge units from the branch channels.

[0048] FIG. 15 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through a spacer and two sets of successive routers, e.g. distributors.

[0049] FIG. 16A provides an illustrative example of a detection system. FIG. 16B provides a photograph of a unit doublet traveling through an optical detection system.

[0050] FIG. 17A-C provide examples of the signal generated by unit singlets (17A), unit doublets (17B), and unit singlets, doublets and multiples (17C) as they pass through the optical detection system of FIG. 16B configured in accordance with the schematics shown in FIG. 16A.

[0051] FIG. 18A-B provides examples of signals generated by single units (18A) and bubbles (18B).

[0052] FIG. 19 provides an illustrative example of a set-up for bead manipulation.

[0053] FIG. 20 provides an illustrative example for a bead mixing mechanism with reagents.

[0054] FIG. 21 provides an image of a double T-junction branch point.

[0055] FIG. 22A-D provides images of a unit stop (A), a unit spacer (B), a unit spacer with polished capillaries inserted (C) and a cross channel unit spacer (D).

[0056] FIG. 23A-D provides snapshots from a movie of beads being separated by a unit spacer.

[0057] FIG. 24A-D provides pictures of (A) a unit stop constructed from a LabSmith union connector, (B) a close up image of the capillary, tubing, and wires in the unit stop of panel (A), (C)-(D) close up images of a wire inserted into a capillary for use as a unit stop with fitting removed, showing wire.

[0058] FIG. 25 provides an image of an exemplary positional encoding device.

[0059] FIG. 26 provides a diagram illustrating exemplary error correction methods and devices in accordance with various embodiments of the invention.

[0060] FIG. 27 provides an illustrative example of a microfluidic device and system comprising a multichannel pressure / flow controller (OB1 Mk3, Elveflow), fluid reservoirs, fluid flow sensors, and automated 2-way valves (LabSmith).

[0061] FIG. 28 depicts a close-up image of an illustrative microfluidic device and system focusing on the pressure controller and reservoirs. Shown are tubing from pressure controller outputs to reservoir caps through a filter and / or a liquid stop 2801, which are pneumatic lines that are configured to pressurize the reservoirs; and 360 um fused silica capillary fluid lines leading from top of reservoirs 2802.

[0062] FIG. 29 depicts an illustrative fluidic breadboard with flow sensors and automated valves. Input fluid lines pass through the flow controllers to the two-way valves. Two-way valves route flow to different parts of the fluidic network. The left valve directs flow to the “top” or “bottom” of a main transport channel (the second channel in FIG. 25 described in further detail elsewhere herein.

[0063] FIG. 30 provides a close-up image of a microfluidic fluid flow sensor (MFS, Elveflow). Top cable is configured to deliver fluid flow data to the multichannel pressure / flow controller depicted in FIG. 27. With the use of the flow sensor, the multichannel pressure / flow controller can be used to perform closed loop control of fluid speed by dynamically adjusting the applied pressure.

[0064] FIG. 31A-F provides diagrams of differential pressure for distributing units in a single T junction.

[0065] FIG. 32A-E provides diagrams of differential pressure for distributing units in a double T junction.

[0066] FIG. 33 provides a close-up image of a cross-flow routing junction. Glass capillaries are inserted into 3D printed routing device. Units, e.g. beads, spaced 40 μm from each other enter from left and a cross-flow current from the vertically oriented capillaries deflect the units into the upper or lower bifurcation.

[0067] FIG. 34A-B provide a schematic (34A) of electroosmotic flow and a close up of an electroosmotic pump (34B). Voltage across two porous electrodes drives electroosmotic flow through a nanoporous pump medium. Electrical leads drive fluidic flow through connected glass capillaries.

[0068] FIG. 35A-C provide close up images on a unit trap (35A), a unit spacer (35B), and a laser detector (35C). FIG. 35A illustrates an exemplary 3D printed unit trap: A disk shaped element with slit in channel is configured to allow fluid flow while preventing units from passing. FIG. 35B illustrates an exemplary unit spacer that is configured to introduce spaces between stacked units. FIG. 35C illustrates an exemplary laser unit detector: 40 μm units, such as beads may be detected by laser as they move left to right in channel.

[0069] FIG. 36 depicts swelling response of divinylbenzene-cross-linked polystyrene (PS) beads in organic solvent versus percent divinylbenzene (DVB) cross-linking agent.

[0070] FIG. 37A-F depict images and schematics of an example device portion for spacing and re-stacking beads without permanent jams. FIG. 37A is a plan view of the example device; FIG. 37B is an elevation view of a schematic of the example device; FIG. 37C is a snapshot of a video of a region of view about a first spacer in the example device;

[0071] FIG. 37D is an image of a detector region of the example device; FIG. 37E is an image of signals produced as beads pass the detector; and FIG. 37F is a snapshot of a video of a region of view about a second spacer in the example device, which when operated in reverse removes or reduces excess fluid between beads and returns them to a more packed configuration.

[0072] FIG. 38 depicts images of an example device portion for steering beads using an in-line spacer element coupled to a Y-channel. Additional, fluidic connections at the Y-channel junction deliver a volume of fluid into the Y-junction, causing beads to be deflected and move into the opposing leg of the bifurcation.

[0073] FIG. 39 depicts an exemplary illustration of a router configured to distribute beads into a plurality of branch channels and / or merge beads from a plurality of branch channels.

[0074] FIG. 40 depicts an exemplary illustration of a router configured to route units into a plurality of branch channels using bubbles generated by a microactuator.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Briefly, and as described in more detail below, described herein are methods and compositions relating to tracking of mobile units within microfluidic devices. Mobile units may be tracked by controlling or recording the relative positioning of the mobile units within the microfluidic device. The tracked mobile units may be distributed into the channels of a microfluidic device, for example by employing a router, such as a distributor, and recombined. The order in which mobile units move through the microfluidic device as they are split into and are recombined from various compartments of the microfluidic device may be controlled and / or recorded. The order or relative position of the mobile units upon recombination may be used to determine the path each mobile unit took through the microfluidic device. Individual channels of the microfluidic device may be used to perform reactions, such as synthesis reactions, e.g. nucleic acid synthesis reactions. Such reactions may be performed in parallel. Reagents for each reaction may be delivered to the individual channels, for example via separate reagent delivery channels. Suitable reaction conditions, such as temperature, pressure, and flow rate may be set in the individual channels. The mobile units may comprise beads such as glass beads, polymer beads, or chemically resistant polymer beads. Synthesis reactions may be performed on a nascent chain on the beads. The mobile units may or may not carry labels or barcodes. The terms ‘label’ and ‘barcode’ shall be used interchangeably herein.

[0076] Provided herein are methods of positionally tracking and moving units within a microfluidic device. The units may be loaded into a microfluidic device. In relation to jamming, clogging, aggregating, and / or keystoning of beads (or other units) at a capillary opening into the microfluidic device, embodiments of the system(s) and method(s) described can implement structures that facilitate loading of beads (or other units) into a capillary in a desired manner.

[0077] In a first embodiment, a first end of a capillary is inserted into a reservoir containing the beads (or other units) in solution, and applying positive pressure to the reservoir to drive beads (or other units), with the solution, into the capillary in a desired manner. Positive pressure can be applied by way of a pump coupled to the reservoir, where the applied pressure can be configured in a manner that controls the rate at which fluid and beads (or other units) are driven into the end of the capillary interfacing with the reservoir.

[0078] Additionally or alternatively, in a second embodiment, a first end of a capillary is inserted into a reservoir containing the beads (or other units) in solution, and a negative pressure (e.g., vacuum) is applied to another region of the capillary (e.g., a second end) to deliver beads (or other units), with the solution, into the capillary in a desired manner. Negative pressure can be applied in a manner that controls the rate at which fluid and beads (or other units) are driven into the end of the capillary interfacing with the reservoir.

[0079] In any of the embodiments of loading described herein, loading beads (or other units) into a capillary can be supplemented with agitation of the vessel or the solution containing the beads (or other units) or by adjusting concentration of the units in solution to modulate bead / unit throughput, and / or to minimize simultaneous bead arrival at the capillary opening(s). For instance, the vessel containing the beads may be affixed to a device that provides any motion including vibration, rocking, shaking, rotation, randomly or periodically. Such motion may be achieved through use of a piezoelectric device, an ultrasound device, or a mechanical mechanism. Additionally or alternatively, fluid containing the beads may be circulated or agitated inside an otherwise stationary vessel using ultrasound, stirrers, stir-bars, paddles, propellers, or additional fluid connections delivering jets of fluids. Additionally or alternatively, a dilution reagent that affects concentration and / or viscosity of the solution can be used to flow beads or units through the device or capillary.

[0080] Additionally or alternatively, in any of the embodiments of loading described herein, the capillary and / or channels fluidly coupled to the capillary upstream of the capillary can be configured with various geometries that reduce the likelihood that multiple beads arrive at the capillary opening simultaneously or in another undesired manner. Such geometries can include changes (e.g., step changes, gradual changes) channel width or diameter, or in wall width or diameter along a downstream-to-upstream direction, which can yield a configuration where the beads or other units are transmitted from the reservoir into the capillary one-by-one. Beads or other units may then be rapidly accelerated into the capillary due to increasing flow rates with smaller cross-sectional area along the flow path into the capillary.

[0081] Additionally or alternatively, in any of the embodiments of loading described above, the system(s) and / or method(s) described herein can be configured to apply and / or take advantage of forces exerted upon the beads or other units opposite to the unit loading direction, thereby controlling unit loading into the capillary. For instance, in embodiments, one or more of: gravitational forces, magnetic forces, electrophoretic forces, dielectrophoretic forces, fluid forces (e.g., using ultrasound, vibration, agitation, or a sheathing stream surrounding a core stream of fluid), and other forces are used alone or in combination with any of the above-described embodiments. Such applications may be used to mitigate or eliminate jamming or a likelihood of simultaneous bead / unit arrival at an opening of the capillary.

[0082] In another such embodiment of a configuration of bead loading using opposing forces, a loading capillary is oriented downward (e.g., parallel to gravitational forces), such that the beads or other units have a natural propensity to flow downward into the capillary due to gravitational forces. Gravity can thus carry the beads or other units, with solution, into the capillary, while an opposing force (e.g., applied negative pressure in an “upward” direction) counters the tendency for units to form aggregates at the opening into the capillary.

[0083] In another such embodiment of a configuration of bead loading using opposing forces, a loading capillary is oriented upward (e.g., parallel to gravitational forces), such that the movement of beads into the capillary is countered by gravitational forces. Suction of units, with solution, into the capillary, while gravity serves as an opposing force counters the tendency for units to form aggregates at the opening into the capillary.

[0084] In various embodiments the opposing forces already described can be applied intermittently, so as to counter or disrupt aggregates that may have formed at the opening into the capilliary. In the case of gravity, which cannot be intermittently applied, the suction (e.g. negative pressure applied to the fluid in the capillary causing beads and fluid to be drawn into the capillary) or positive pressure (e.g. positive pressure inside the vessel that causes beads and fluid to move into the opening of the capillary) may be intermittently reduced or stopped, so counter or disrupt aggregates that may have formed at the opening of the capillary.

[0085] In various embodiments, channels or other fluidic structures fluidly coupled to the capillary can include grooves or similar structures that facilitate formation of rows of units prior to entering the opening of the capillary mitigating likelihood of aggregation at the opening. In variations, however, the capillary may not be oriented upward or downward (e.g., parallel to gravitational forces) and can alternatively be oriented at any angle between vertically upward or vertically downward.

[0086] In various embodiments, the systems described herein comprise elements that apply opposing magnetic forces (e.g., for magnetic units) and / or opposing electric forces (e.g., for charged units) or other suitable types of forces described herein or known in the art. Such forces can be used to control flow of beads or other units into a loading channel, e.g. a loading capillary. For instance, adjustment of a net force applied to each unit (e.g. bead) as it enters a capillary, such as through opposing electromagnets or permanent magnets that are placed at an adequate distance relative to the capillary, can be used to control position, velocity, and / or acceleration of unit(s) (e.g. beads) into and within the capillary in a manner that prevents jamming or other undesired transmission.

[0087] Additionally or alternatively, in another such embodiment, a Bernoulli force applied across a second end of the capillary away from a first end of the capillary can be used to generate a pressure reduction that promotes transmission of the bead(s) or other units into the capillary in a desired manner. The system can apply a Bernoulli force through structures providing crossflow of fluid across the second end of the capillary.

[0088] In various embodiments, unit loading channels are oriented directly parallel or directly opposite forces applied to units in solution prior to loading, or at any other suitable angle. Forces applied to units in pre-loading solution may be adjusted such that the component of the force along the direction of the flow has a suitable absolute or relative value (e.g. relative to the pressure differential for flow into the loading channel) that can mitigate or eliminate aggregation at the entrance of the loading channel.

[0089] Provided herein are also methods of spacing or ejecting units within a microfluidic device. Provided herein are methods of steering or distributing units within a microfluidic device. Provided herein are methods of trapping or holding units within a microfluidic device. Provided herein are methods of tracking units within a microfluidic device. Provided herein are methods of dispensing units within a microfluidic device.

[0090] Provided further herein are methods to prototype fluidic components and networks including micromachining, soft lithography with polydimethylsiloxane (PDMS), 3D printing, and photolithography. In various embodiments, such methods are used to perform fundamental operations for microfluidic devices and systems described herein (e.g. a desktop synthesizer), including without limitation operations such as moving, stacking, spacing, steering, and counting units, e.g. beads. In various embodiments, stacking of units is achieved by tightly packing units in the microfluidic devices described herein. In some embodiments, stacking is used to allow for efficient application of reaction conditions or treatments (e.g. those aimed to achieve desired chemical reactions) in small reagent volumes. In some embodiments, spacing of units during various routing operations is used to enable deterministic deflection of individual units, e.g. beads. In various embodiments, a method for optical detection is used in order to reliably detect and count units at one or multiple points within the device, allowing for accurate tracking of units some or all times, even in the event that a unit is incorrectly steered.

[0091] In further embodiments, use of highly spherical and uniformly sized units allows for thousands of such to be driven as a one-dimensional (1D) array through narrow channels, for example by application of fluidic pressures. In some embodiments, such units in a 1D array hundreds of times in succession are driven through the microfluidic devices described herein without jamming, clogging, or fragmenting.

[0092] It must be noted that, as used in the specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearly dictates otherwise.

[0093] Using phosphoramidite DNA synthesis chemistry molecules can be synthesized on the surface of a solid support substrate in a step-by-step reaction proceeding, generally, in the 3′ to 5′ direction and consisting of (1) a detritylation step to remove a protecting group from the previously added nucleoside (this prevents more than one nucleoside from being added per cycle), (2) a coupling of the next nucleoside to the growing DNA oligomer, (3) oxidation to convert the phosphite triester intermediate into a more stable phosphate triester, (4) irreversibly capping any unreacted 3′ hydroxyls groups. Without being bound by theory, capping unreacted 3′ hydroxyl groups can help prevent synthesized sequences having a deletion relative to preselected nucleic acid sequences by avoiding continued polymerization from such 3′ hydroxyl groups in subsequent cycles. The cycle can be repeated to add the next base. Solid supports may comprise a variety of units, such as beads, including without limitation highly porous polymeric beads; glass or silica beads including, but not limited to fused silica (amorphous pure silica), quartz (crystalline pure silica); or other any other suitable beads described herein or otherwise known in the art, which can be packed into a chamber or column, to which the synthesis reagents are delivered. The methods, devices and compositions described herein can be used to scale nucleic acid synthesis methods using microfluidic approaches.

[0094] Microfluidic approaches can be used to for applications of solid phase phosphoramidite chemistry. In some embodiments, mobile solid support units are delivered to one of four chambers in each cycle of an iterative process. In this approach, mobile units to be extended with a particular nucleoside may be delivered and comingled to the same chamber on that particular cycle. After each cycle the units may be redistributed to be delivered again to the appropriate chamber to receive the next base. In some embodiments, units are selected from beads having a diameter and / or size in the range of 10-100 μm. The beads may be monodisperse. Nucleic acids may be synthesized on a plurality of units, including without limitation beads, for example on ten to ten thousand beads or on hundreds of thousands to millions of beads in parallel in a small microfluidic device. Implementation of this approach may comprise one or more of (1) a set-up for encoding hundreds of thousands to millions of units, such as 10-100 μm beads, with of unique barcodes, (2) a set-up for detecting the units while beads are moving at high speeds, (3) a method for directing or distributing beads into the appropriate output chambers on each iteration, and (4) integration of these components in a functional microfluidic system for iterative operation.

[0095] In some embodiments, oligonucleotides are synthesized in the 5′ to 3′ direction. In some embodiments, 5′ to 3′ synthesis is achieved by performing one or more of (i) functionalizing of units; e.g. silanization, amino functionalization, hydroxyl functionalization; (ii) providing photolabile 5′-phosophoramidites, e.g. 3′-NPPOC-deoxyadenosine (N6-benzoyl)-5′-β-cyanoethylphosphoramidite, 3′-NPPOC-deoxycytidine (N4-acetyl)-5′-β-cyanoethylphosphoramidite, 3′-NPPOC-deoxyguanosine (N2-dimethylformamidine)-5′-β-cyanoethylphosphoramidite and / or 3′-NPPOCdeoxythymidine-5′-β-cyanoethylphosphoramidite; (iii) dosing the units with light of a suitable wavelength, e.g. UV light; and (iv) coupling a photolabile 5′ phosphoramidite to the functionalized unit and / or to a nascent oligonucleotide associated with the unit. Oxidation, capping and deprotection steps may be performed similar to 3′ to 5′ phosphoramidite synthesis. (See e.g. Albert et al., Nucleic Acids Research, 2003, Vol. 31, No. 7 e35, DOI: 10.1093 / nar / gng035; Nuwaysir et al., Genome Res. 2002. 12:1749-1755, doi:10.1101 / gr.362402; and Singh-Gasson et al., Nature Biotechnology volume 17, pages 974-78 (1999)).

[0096] In some embodiments, 5′ to 3′ synthesis is achieved by performing one or more of (i) functionalizing of units; (ii) providing phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group (reverse BzNPPOC phosphoramidites); (iii) performing a coupling reaction with the reverse BzNPPOC DNA phosphoramidites, e.g. by irradiating the units with light of appropriate wavelength, e.g. UV light; (iv) performing a capping step; and (v) performing an oxidizing step. Deprotection steps may be performed prior to the addition of new reverse BzNPPOC phosphoramidites. (See e.g. Holz et al., Scientific Reports (2018) 8:15099, DOI:10.1038 / s41598-018-33311-3.)

[0097] Forward and reverse phosphoramidites (i.e., phosphoramidites with a protecting group in the 5′ or 3′ position, respectively) may be purchased from Glen Research, Sterling, Virginia.

[0098] Since microbead barcoding problem had thwarted a number of groups and prevented development of a working device, innovative alternative technology was developed. In various embodiments, methods and compositions described herein comprise a fluidic device in which the beads or other types of units are constrained to narrow fluidic channels, such that they are maintained in a one-dimensional array (FIG. 1-3). This system, in various embodiments, allows the beads or other types units to be identified by their position alone. In some embodiments, beads are loaded into a primary channel. The primary channel may be a capillary or a channel engineered into a suitable substrate. As the beads or other types of units begin the process in a primary channel, they can be pushed, one-by-one, through a distributing mechanism that would direct the beads or other types of units into an appropriate branch channel. Both the primary channel and branch channels may be sized to prevent the beads sliding or squeezing past one another. Once distributed, phosphoramidite chemistry, or other desired chemistry, can take place in the branch channel. After completion of each round of synthesis, the beads or other types of units may be moved in an ordered fashion back into the primary channel for redistributing and a subsequent round of synthesis. In some embodiments, the diameter and / or size of the units and corresponding channels is configured such that units cannot pass each other within a channel or would do so at a rate that is lower than a threshold. For example, units having a diameter and / or size greater than 50% of the width of the channel containing them may be selected.

[0099] A T-junction or flow focusing method may be configured to eject beads or other types of units from the terminus of the primary array and move them towards a router, e.g. a distributor, for example one at a time. Introducing a gap between units may allow for optical detection and routing, e.g. distribution, before the next unit reaches the router. The router may direct the units into one of the branch channels and / or reaction chambers. One or more of available branch channels or reaction chambers may be configured to allow addition of one of the four DNA bases to a nascent nucleic acid, e.g. DNA chain. The distributing mechanism may potentially comprise a multiway router, or a router with two sequential binary routers enabling multiple branchings (FIG. 4). A set of optical detectors could be positioned at the outlets of one or more routers to verify that each unit was distributed to the intended outlet. Once some or all the units have been distributed, a cycle of the phosphoramidite chemistry may be performed in some or all of the branch channels or reaction chambers and an appropriate nucleoside may be added to nucleic acid molecules in or on some or all the units in some or all of the branch channels or reaction chambers. A subsequent cycle can involve a different chemistry, e.g., addition of modified nucleosides or non-phosphoramidite nucleosides, or treatment e.g., a physical or light based treatment. The methods and devices described herein may be used to apply a different reaction or treatment to some or all branch channels or reaction chambers in some or all cycles. In some embodiments, units are redistributed between cycles of reactions or treatments. The cycles of reactions or treatments may be asynchronous for the units held in different branch channels or reaction chambers. For example, if units are held in two or more branch channels, units held in one branch channel may undergo a first cycle of reaction, and subsequently all of the units held in some or all of the branch channels may undergo a second cycle reaction.

[0100] Introduction of the synthesis reagents may be accomplished by using separate reagent ports, e.g. near the beginning of branch channel or reaction chamber outlets. After the completion of a round of synthesis, the units may be recirculated in an ordered fashion back into the primary channel for redistributing and a subsequent round of synthesis. In some embodiments, such recirculation of units comprises reversing the direction of the units backward relative to the direction units entered a branch channel or reaction chamber, thereby causing the units to move into a primary (or main) channel. The process may be repeated as desired, e.g. until the nucleic acid synthesis on all units is complete. In some embodiments, a fluidic device includes an additional output channel to enable synthesis of nucleic acids, e.g. DNA sequences, of different lengths. As modification, e.g. synthesis, on a unit is completed, it may be directed to such an additional output channel and be kept from cycling through the process further. Additional routers, e.g. distributors, and / or sub-channels may be used to handle units that have been incorrectly distributed. Such routers and / or sub-channels may be used to redirect units for redistributing into a correct channel immediately, or directing them into channels where no modifications are made, and then moving these units back into the primary channel before the next cycle so they can be distributed correctly.

[0101] This approach can circumvent the need for a barcoding technology entirely. It can also eliminate the need for a complex and potentially expensive optical detection and image processing system. Instead of a costly system, simple optical detectors may be optionally implemented for counting beads. In various embodiments, beads and other types of units may be processed at high speeds. Further, low cost optical checkpoints may be implemented to verify correct distributing.

[0102] In various embodiments, the order of the mobile units as they are routed within the microfluidic devices described herein is set in a deterministic manner, for example by distributing and releasing the units into and from reaction chambers in a predetermined manner. The position or relative position of specific mobile units may be known or determinable from the path each mobile unit has taken in a prior round of distributing and recombining. In some embodiments, the order of the units is set by tracking the units by detectors operably connected to detect units as they are routed within the microfluidic devices described herein. The devices and methods described herein allow for positional encoding, such that the order of mobile units within the device at a given time and / or location carries information about the path a unit has followed during routing steps. For example, the order of units may be used to determine, which of a plurality of branch channels a unit has been distributed to and / or merged from. In some embodiments, information that was used to determine the order of the beads, such as tracking information, is itself determinative of elements of the routing path that a unit has been routed through. In some embodiments, the devices and methods described herein are configured to route units through a microfluidic device deterministically. The order of units at a given time and / or location within the microfluidic devices described herein, in combination with such a routing algorithm may be used to determine elements of the routing path that a unit has been routed through.

[0103] Elements of a unit's routing path may be determinative of the identity of a compound that was synthesized on a unit as it was routed through the microfluidic devices described herein. More generally, the reaction conditions and / or treatments to which a unit has been subjected to as it was routed through the microfluidic devices described herein, as well as their order, may be determined from the location of the unit. In various embodiments, such location relates to a relative position of a unit within an ordered set of units. Units that have been routed through the microfluidic devices described herein may be mapped to specific routing paths with the use of position information specified relative to other units within the microfluidic device, such as units that are in close vicinity of a particular unit within an ordered set of units.

[0104] In various embodiments, chemical products may be associated with mobile units. The chemical compounds may be in or on the mobile units, they may be tethered or attached, or adsorbed by the mobile unit. The units may be identified by their positional relationship either to each other or to the system. The chemical products associated with each unit may be determined by the history modification procedures applied to each unit. In various embodiments, the absolute or relative position of the units is controlled over time. The positional relationship of the mobile units may be controlled by a variety of suitable methods. For example, the positional relationship may be maintained by ordering the units, for example in a one-dimensional array (1d-array; e.g. single row). This array of units can be split into two or more new branch arrays, which may be one dimensional. The direction of the unit flow through the splits may be controlled. The positional information of the units may be updated with each split. The positional information may include both the new branch array assignment and the position within the new branch array. The various branch arrays comprising the units may be subjected to different modification procedures. A modification procedure may be applied to all of the units in a branch array. The modification procedures and the order of application for modification procedures for each unit may be recorded. After performing modification procedures on the branch arrays, the units in the two or more branch arrays may be merged into a single array. The merging of branch arrays can also be controlled such that the order, branch array history, position, and any procedures applied to units in the new array is recorded. This information may be captured and stored in a computer memory using software specifically built for this purpose. The method may consist of any number of splits, modification procedures, and mergers of branch arrays, wherein the position of and the history of the applied procedures for the units are controlled. The units may be moved through splits, branch arrays, and mergers in series, in parallel, in a loop, or a combination thereof. A large number units, e.g. about, more than, or more than about 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more units, can be directed in a deterministic fashion, having a large number of independent modification procedures applied to produce large targeted or combinatorial libraries of products on the mobile units. Values for the number of units may range between any of the potential values set forth for the number of unit herein. In some embodiments, units are directed through the channels of a microfluidic device without specifically controlling the path for each unit or randomly. Such units may also be tracked and thereby positionally encoded, for example based on the units' relative positions. Tracking information can be used to determine the chemical steps a unit has gone through, for example in split synthesis applications. The products on each unit may be predicted or determined based on the chemical steps the unit has gone through.

[0105] The branch arrays and corresponding modification procedures to be applied to units flowing through may be specifically pre-assigned at every split such that some or all units receive a specific set of modification procedures and are directed appropriately at each splitting event. The series of modifications may be preordained, but assigned to units randomly. In some embodiments, the series of modifications is not preordained. The units may be assigned to a series of modifications deterministically or randomly, e.g. every other unit or an average 50% of units may be directed to a certain path during the splitting event. Regardless of how the assignments are made, the position of units and modification procedures may be recorded.

[0106] Suitable designs for the system and units may be selected in order to enable or enhance features of the methods and compositions relating to the invention. For example, ratios between unit size, height, length, width, diameter, and / or cross-section and / or fluidic channel size, height, width, depth, diameter, and / or cross-section may be selected such that the units would not typically be disarranged or mixed under routine operating conditions, thus maintaining the order of the units within a channel, including without limitation in narrow channels physically restricting mixing or as units are moved within channels in maintained order, for example in in laminar or laminar-like flow. The units can be directed from a single channel, into two or more branch channels by any appropriate mechanism, such as pressure differential, flow focusing (e.g., hydrodynamic focusing), lateral movement of the unit in the laminar flow, valves, gates, routers described in further detail herein, e.g. distributors, or switches of various types (e.g. acoustic, electrophoretic, or photonic) and / or other suitable mechanisms known in the art. Flow focusing may include acoustic focusing and inertial focusing, as described in further detail in U.S. Pat. Nos. 7,340,957 and 9,347,595, both of which are herein incorporated by reference in their entirety. The force inducing the movement of the units through the channels may be from fluidic pressure created by a pump, from electroosmotic forces, or any other transport mechanism known in the art. The input channel or the branch channel, or other channels described in further detail elsewhere herein may be associated with a detector. The detector may be configured to count units, confirm that units were directed into the correct channel, or otherwise track the units and / or the units' relationship to each other or to fiducial marks in the microfluidic device. In some embodiments, units are reordered based on detector read-out, for example when units are erroneously distributed. The detector(s) may be coupled to programs, such as computer programs on a computer configured to accept input from the detector(s). Based on the input from the detector(s), for example when the detector detects certain features, the program may execute certain functions. For example, the detector(s) may be coupled to a feedback loop, such as a feedback loop for controlling the pressure of pumps within or coupled to a microfluidic device. The pressure control may be used to control / adjust the speed of the units. The direction or speed of clumped or adhered units may be adjusted. For example, units may be directed into a particular channel so that they can be separated or isolated from the remainder of the units. Detectors of any suitable type may be used in various embodiments of the invention, including without limitation laser or LED detectors, or CCD based devices. Two or more channels, such as branch channels, may converge into one output path. The movement of the units may be controlled and / or positions of the units in the output channel may be updated as the units are combined in the output path. In one embodiment, units from multiple channels may be merged into a single channel by directing units from one channel through a merging branch point first and subsequently directing the units from a second channel through the merging branch point. The absolute or relative positions of some or all of the units may be tracked or determined accordingly.Channels

[0107] Within a microfluidic system designed to hold ordered sets of, for example channels sized to hold i d-arrays of units, the capacity of the channel may be set based on the average diameter, size, or cross-section of the units. The channels may be narrow to physically constrain the units as they move through the channel such that a unit cannot physically pass the unit ahead or behind it. For example, the channel width may be between 1 to 2 times the average or nominal diameter and / or size of the units. In some embodiments, units are constructed of a rigid non-compliant material, such as glass or rigid polymer, e.g. polystyrene crosslinked with divinyl benzene, or other suitable polymer know in the art. In some embodiments, units constructed from rigid non-compliant material are held or flowed in the microfluidic channels described herein. Units constructed from such rigid non-compliant material may be maintained in order by physically preventing them from passing each other inside channels that are narrow enough to constrict them. Channels may be broad enough to allow for the passage of units constructed from rigid non-compliant materials. In some embodiments, the ratio of average or nominal unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, more than, or more than about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or more. In some embodiments, the ratio of average or nominal unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, less than, or less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55 or less. In some embodiments, the ratio of unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel falls within a range bounded by any of the foregoing values, for example 0.45-0.99, 0.45-0.95, 0.45-0.90, 0.45-0.85, 0.45-0.80, 0.45-0.75, 0.45-0.7, 0.45-0.65, 0.45-0.6, 0.5-0.99, 0.5-0.95, 0.5-0.90, 0.5-0.85, 0.5-0.80, 0.5-0.75, 0.5-0.7, 0.5-0.65, 0.5-0.6, 0.5-0.55, 0.55-0.99, 0.55-0.95, 0.55-0.90, 0.55-0.85, 0.55-0.80, 0.55-0.75, 0.55-0.7, 0.55-0.65, 0.55-0.6, 0.6-0.99, 0.6-0.95, 0.6-0.90, 0.6-0.85, 0.6-0.80, 0.6-0.75, 0.6-0.7, 0.6-0.65, 0.6-0.6, 0.65-0.99, 0.65-0.95, 0.65-0.90, 0.65-0.85, 0.65-0.80, 0.65-0.75, 0.65-0.7, 0.65-0.65, 0.7-0.99, 0.7-0.95, 0.7-0.90, 0.7-0.85, 0.7-0.80, 0.7-0.75, 0.75-0.99, 0.75-0.95, 0.75-0.90, 0.75-0.85, 0.75-0.80, 0.8-0.99, 0.8-0.95, 0.8-0.90, 0.8-0.85, 0.85-0.99, 0.85-0.95, or 0.85-0.90. Values for the channel ratio may range between any of the potential values set forth for the channel ratio herein.

[0108] In some embodiments where units are constructed from a compliant material, such as droplets, slugs, immiscible volumes, hydrogels, or compliant polymers, the ratio of average or nominal uncompressed unit diameter and / or size (as measured outside of the channel) to channel width may be substantially larger than 1. In some embodiments, the ratio of average or nominal uncompressed unit diameter and / or size (e.g. as measured outside of the channel) to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, more than, or more than about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.30, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.70, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.5, 3.0, 3.5, 4.0 or more. In some embodiments, the ratio of average or nominal uncompressed unit diameter and / or size the ratio of average or nominal uncompressed unit diameter and / or size (as measured outside of the channel) to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, less than, or less than about 4.0, 3.5, 3.0, 2.5, 2.0, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.90, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 055 or less. In some embodiments, the ratio of unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel falls within a range bounded by any of the foregoing values, for example 0.5-4, 0.5-3.5, 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.95, 0.5-1.85, 0.5-1.8, 0.5-1.75, 0.5-1.7, 0.5-1.65, 0.5-1.6, 0.5-1.55, 0.5-1.5, 0.5-1.45, 0.5-1.4, 0.5-1.35, 0.5-1.3, 0.5-1.25, 0.5-1.2, 0.5-1.15, 0.5-1.1, 0.5-1.05, 0.5-1, 0.5-0.95, 0.5-0.9, 0.5-0.85, 0.5-0.8, 0.5-0.75, 0.5-0.7, 0.5-0.65, 0.5-0.6, 0.5-0.55, 0.55-4, 0.55-3.5, 0.55-3, 0.55-2.5, 0.55-2, 0.55-1.95, 0.55-1.85, 0.55-1.8, 0.55-1.75, 0.55-1.7, 0.55-1.65, 0.55-1.6, 0.55-1.55, 0.55-1.5, 0.55-1.45, 0.55-1.4, 0.55-1.35, 0.55-1.3, 0.55-1.25, 0.55-1.2, 0.55-1.15, 0.55-1.1, 0.55-1.05, 0.55-1, 0.55-0.95, 0.55-0.9, 0.55-0.85, 0.55-0.8, 0.55-0.75, 0.55-0.7, 0.55-0.65, 0.55-0.6, 0.5-0.55, 0.6-4, 0.6-3.5, 0.6-3, 0.6-2.5, 0.6-2, 0.6-1.95, 0.6-1.85, 0.6-1.8, 0.6-1.75, 0.6-1.7, 0.6-1.65, 0.6-1.6, 0.6-1.55, 0.6-1.5, 0.6-1.45, 0.6-1.4, 0.6-1.35, 0.6-1.3, 0.6-1.25, 0.6-1.2, 0.6-1.15, 0.6-1.1, 0.6-1.05, 0.6-1, 0.6-0.95, 0.6-0.9, 0.6-0.85, 0.6-0.8, 0.6-0.75, 0.6-0.7, 0.6-0.65, 0.65-4, 0.65-3.5, 0.65-3, 0.65-2.5, 0.65-2, 0.65-1.95, 0.65-1.85, 0.65-1.8, 0.65-1.75, 0.65-1.7, 0.65-1.65, 0.65-1.6, 0.65-1.55, 0.65-1.5, 0.65-1.45, 0.65-1.4, 0.65-1.35, 0.65-1.3, 0.65-1.25, 0.65-1.2, 0.65-1.15, 0.65-1.1, 0.65-1.05, 0.65-1, 0.65-0.95, 0.65-0.9, 0.65-0.85, 0.65-0.8, 0.65-0.75, 0.65-0.7, 0.7-4, 0.7-3.5, 0.7-3, 0.7-2.5, 0.7-2, 0.7-1.95, 0.7-1.85, 0.7-1.8, 0.7-1.75, 0.7-1.7, 0.7-1.65, 0.7-1.6, 0.7-1.55, 0.7-1.5, 0.7-1.45, 0.7-1.4, 0.7-1.35, 0.7-1.3, 0.7-1.25, 0.7-1.2, 0.7-1.15, 0.7-1.1, 0.7-1.05, 0.7-1, 0.7-0.95, 0.7-0.9, 0.7-0.85, 0.7-0.8, 0.7-0.75, 0.75-4, 0.75-3.5, 0.75-3, 0.75-2.5, 0.75-2, 0.75-1.95, 0.75-1.85, 0.75-1.8, 0.75-1.75, 0.75-1.7, 0.75-1.65, 0.75-1.6, 0.75-1.55, 0.75-1.5, 0.75-1.45, 0.75-1.4, 0.75-1.35, 0.75-1.3, 0.75-1.25, 0.75-1.2, 0.75-1.15, 0.75-1.1, 0.75-1.05, 0.75-1, 0.75-0.95, 0.75-0.9, 0.75-0.85, 0.75-0.8, 0.8-4, 0.8-3.5, 0.8-3, 0.8-2.5, 0.8-2, 0.8-1.95, 0.8-1.85, 0.8-1.8, 0.8-1.75, 0.8-1.7, 0.8-1.65, 0.8-1.6, 0.8-1.55, 0.8-1.5, 0.8-1.45, 0.8-1.4, 0.8-1.35, 0.8-1.3, 0.8-1.25, 0.8-1.2, 0.8-1.15, 0.8-1.1, 0.8-1.05, 0.8-1, 0.8-0.95, 0.8-0.9, 0.8-0.85, 0.85-4, 0.85-3.5, 0.85-3, 0.85-2.5, 0.85-2, 0.85-1.95, 0.85-1.85, 0.85-1.8, 0.85-1.75, 0.85-1.7, 0.85-1.65, 0.85-1.6, 0.85-1.55, 0.85-1.5, 0.85-1.45, 0.85-1.4, 0.85-1.35, 0.85-1.3, 0.85-1.25, 0.85-1.2, 0.85-1.15, 0.85-1.1, 0.85-1.05, 0.85-1, 0.85-0.95, 0.85-0.9, 0.9-4, 0.9-3.5, 0.9-3, 0.9-2.5, 0.9-2, 0.9-1.95, 0.9-1.85, 0.9-1.8, 0.9-1.75, 0.9-1.7, 0.9-1.65, 0.9-1.6, 0.9-1.55, 0.9-1.5, 0.9-1.45, 0.9-1.4, 0.9-1.35, 0.9-1.3, 0.9-1.25, 0.9-1.2, 0.9-1.15, 0.9-1.1, 0.9-1.05, 0.9-1, 0.9-0.95, 0.95-4, 0.95-3.5, 0.95-3, 0.95-2.5, 0.95-2, 0.95-1.95, 0.95-1.85, 0.95-1.8, 0.95-1.75, 0.95-1.7, 0.95-1.65, 0.95-1.6, 0.95-1.55, 0.95-1.5, 0.95-1.45, 0.95-1.4, 0.95-1.35, 0.95-1.3, 0.95-1.25, 0.95-1.2, 0.95-1.15, 0.95-1.1, 0.95-1.05, 0.95-1, 1-4, 1-3.5, 1-3, 1-2.5, 1-2, 1-1.95, 1-1.85, 1-1.8, 1-1.75, 1-1.7, 1-1.65, 1-1.6, 1-1.55, 1-1.5, 1-1.45, 1-1.4, 1-1.35, 1-1.3, 1-1.25, 1-1.2, 1-1.15, 1-1.1, or 1-1.05. Values for the channel width and / or channel ratio may range between any of the potential values set forth for the channel width and / or channel ratio herein.

[0109] The units may be flowed from or to areas where positional ordering is maintained by a physical dimensional constraint, as described in further detail elsewhere herein, into, through, or from portions of the device not having constricting dimensions for physically constraining mixing of units. However, ordered flow of units may be maintained under suitable operating conditions, such as by the application of laminar or laminar-like flow. Operating conditions for maintaining positional order may be maintained at all times, or some of the times, during operation of the device. In some embodiments, microfluidic devices described herein have areas of expansions, gradual or abrupt, in the channel width in some or all directions, for example, a narrow channel with a circular cross section transitioning to a channel with a rectangular cross section and a wide aspect ratio. Such expansions may increase one or more dimension of a channel such that mixing of units flowing therein is not constrained by the physical dimensions of the channel. Such areas of expansions may also include corners and / or chambers of various aspect ratios. Without being bound by theory, in laminar or streamline, flow, parallel layers of fluid flow without disruption between the layers. Positional ordering of units may be maintained as the units are moved through an expansion, by moving the units in ordered flow, such as in laminar or laminar-like flow conditions sufficient to maintain ordering of units. Flow in such expansions need not necessarily be laminar, but maintenance of positional ordering may be established by adjusting flow conditions empirically, in accordance with the various embodiments herein. In various embodiments, devices and methods described herein maintain ordered flow of units, including without limitation while moving units in less than perfect laminar flow or while holding beads, for example as limited by the rate of diffusion. In various embodiments, units are flowed from a first area of the device where position is maintained via physical constraints, as described, into a second area, where order can be maintained by the application of suitable fluidic conditions during the operation of the devices described herein. For example, in such a second area, the channel cross-section width at its widest point may be between 2 to 1000 times the average diameter and / or size of the units. The channel cross-section width at its widest point may be about, more than, or more than about 2, 2.2, 2.4, 2.5, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 500, 1000 or more times the average or nominal diameter and / or size of the units. The channel cross-section width in its widest dimension may fall within a range bounded by any of the foregoing values, including for example 2-2.5, 2-4, 2.5-3, 2-5, 3-3.5, 3.5-4, 3.5-5, 4-4.5, 4.5-5. 5-10, 10-25, 25-50, 50-75, or 75-100, 100-200, 200-500, 500-1000 times the nominal or average diameter and / or size of the units. Units may be moved further into a third area of the device having constricting dimensions allowing for maintaining the order of units physically. In various embodiments, units are held in a designated order in channels that expand and / or constrict. For example, units held in a channel having a sufficiently small width to physically constrict unit mixing may be moved into another region of the channel or another channel having greater width in at least one dimension, such as a width that is about, is more than, or is more than about 2, 2.2, 2.4, 2.5, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 500, 1000 or more times the average or nominal diameter and / or size of the units. Units in such an expanded region of a channel may be kept in a designated order, for example by keeping units in laminar flow. Similarly, units kept in designated order within a region of a channel that is too wide for physically constricting mixing may be moved into another region of the channel or another channel having a width that is narrow enough to physically constrict mixing, for example a channel width that is about, is less than, or is less than about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.02, 1.01, or 1 times the average or nominal diameter and / or size of the units therein. Such channel widths may be about, less than, or less than about 0.99, 0.95, 0.9, 0.8, 0.7, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1 times the uncompressed (e.g. as measured outside of the channel) average or nominal diameter and / or size of the units therein or less and may still be able to flow compressible or compliant units through. Such channel width transitions may occur in a transition length that is about, is less than, or is less than about 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, or less. Values for the channel width transitions may range between any of the potential values set forth for the channel width transitions herein.

[0110] In some embodiments, the channel width or mean channel width is or is greater than 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1000 μm or greater. In some embodiments, the channel width or mean channel width is or is less than 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, or less. Channels of the devices described herein may have a channel width or mean width within a range bounded by any of the dimensions listed herein, for example 1-5 μm, 3-8 μm, 5-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 1-100 μm, 100-200 μm, 200-300 μm, 300-400 μm, 400-500 μm, or 100-500 μm, 500-1000 μm. In some embodiments, the height to width aspect ratio of the channel(s) can be 1:100 or greater, e.g. 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:19, 1:18, 1:17, 1:16, 1.15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1.5, 1:4, 1:3, 1:2, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, or greater. The height to width aspect ratio can also be less than 1:1, e.g. less than 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or less. In some embodiments, the height to width aspect ratio of the channel(s) can be 10:1 or less, e.g. 100:1, 90:1, 80:1. 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1 or less. The height to width aspect ratio can also be greater than 1:1, e.g. greater than 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1 80:1, 90:1, or 100:1 or more. The height to width aspect ratio of a channel may fall within a range bounded by any of the values listed above, for example the height width aspect ratio may be between 1:100 and 1:20, 1:20 and 1:1, 1:1.1 and 1.5:1, or 1:3 and 3:1.

[0111] The channel(s) length(s) can be about, greater than, or greater than about 0.01 millimeter (mm), 0.1 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 90 mm, 100 mm, 15 centimeters (cm), 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 90 cm, 100 cm, 1.5 meter (m), 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, or more. The channel lengths may fall in a range bounded by any of the dimensions listed herein, e.g. within 1-10 mm, 10-15 mm, 15-20 mm, 20-25 mm, 30-35 mm, 35-45 mm, 45-50 mm, 50-55 mm, 55-60 mm, 60-65 mm, 65-70 mm, 70-75 mm, 75-80 mm, 80-90 mm, 90-100 mm, 10-15 cm, 15-20 cm, 20-25 cm, 30-35 cm, 35-45 cm, 45-50 cm, 50-55 cm, 55-60 cm, 60-65 cm, 65-70 cm, 70-75 cm, 75-80 cm, 80-90 cm, 90-100 cm, 1-2 m, 2-3 m, 3-4 m, 4-5 m, 5-6 m, 6-7 m, 7-8 m, 8-9 m, 9-10 m. The channel(s) length(s) can be about, less than, or is less than about 10 m, 9 m, 8 m, 7 m, 6 m, 5 m, 4 m, 3 m, 2 m, 100 cm, 90 cm, 80 cm, 70 cm, 60 cm, 50 cm, 40 cm, 30 cm, 20 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.01 mm, or less. Values for the channel length may range between any of the potential values set forth for the channel length herein.

[0112] In some embodiments, the length of one or more channels is selected based on the number of units in the device or the number of units that are designated to fit in the channel. Unit sizes are described in more detail elsewhere herein including without limitation in the Unit section in paragraph 129. The channel length may be selected to fit a number of units in a range bounded by any of the values listed herein, e.g., about 1-1E7 units, 1-10, 10-50, 50-100, 50-1E5, 100-500, 100-5E5, 100-1E7, 500-1E4, 1E4-5E4, 5E4-1E5, 1E5-5E5, 5E5-1E6, 1E6-5E6, or 5E6-1E7 units. The channel length may be selected to fit about, more than, or more than about 1, 10, 50, 100, 500, 1E4, 5E4, 1E5, 5E5, 1E6, 5E6, 1E7, or more units. A channel length may be selected to fit about, less than or less than about 1E7, 5E6, 1E6, 5E5, 1E5, 5E4, 1E4, 500, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 units. A branch channel length may be selected to fit a number of units in a range bounded by any of the values listed herein, e.g., 1-1E7 units, 1-10, 10-50, 50-100, 50-1E5, 100-500, 100-5E5, 100-5E7, 500-1E4, 1E4-5E4, 5E4-1E5, 1E5-5E5, 5E5-1E6, 1E6-5E6, or 5E6-1E7 unit lengths. A branch channel length may be selected to fit about, less than, or less than about 1E7, 5E6, 1E6, 5E5, 1E5, 5E4, 1E4, 500, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 units. A branch channel length may be selected to fit about, more than, or more than about 1, 5, 10, 20, 30, 40 50, 100, 500, 1E4, 5E4, 1E5, 5E5, 1E6, 5E6, 1E7, or more units. Values for the branch channel length may range between any of the potential values set forth for the branch channel length herein.

[0113] The units may be spaced from each other with spacer lengths about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the nominal or average size and / or diameter of the units or more. The channel(s) may be selected to have sufficient length to accommodate a desired number of units, for example 1-1E7 units with a spacer length of about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the length of a unit between each unit or more. The channel(s) may be selected to have sufficient length to accommodate 1-1E7 units with a spacer length of about, less than, or less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the length of a unit between each unit or less. The channel(s) may be selected to have sufficient length to accommodate 1-1E7 units with spacer lengths falling within a range bounded by any of the spacer length values described herein, for example 1-1000, 1-100, 2-25, 3-40, 4-10, 5-100, 6-30, 7-100, 8-100, 9-10, 10-15, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 spacer length between each unit. Values for the spacer length may range between any of the potential values set forth for the spacer length herein.

[0114] The channel cross-section shape may be square, rectangle, oval, circular, half-circular, or any other suitable shape. Microfluidic channels can be linear, serpentine, or have another suitable shape or length to enable channels with large unit capacities. Unit capacities of 1E6, 1E7 or higher may be achieved using suitable channel configurations on relatively small fluidic chips.

[0115] According to the various embodiments, channels can be used as reaction chambers where modification procedures are used to modify the products, or in some cases the units. Modification procedures may comprise any chemical, physical, optical, or mechanical method. Various embodiments of the invention ensure that modification procedures do not interfere with the arrangement of the units. Chemical reagents may be flowed as liquids or gasses through the fluidic channel(s) containing the units. The characteristics or diameter and / or size of the channel or the units may be selected to enhance the flow of chemical reagents, or the effectiveness or efficiency of chemical procedures. For example, the channels may be constructed from glass, chemically resistant polymers, or non-resistant polymers or coated with the same. In various embodiments, the channels are chemically resistant to the modification procedures applied. Units may be constructed from any suitable material, such as controlled pore glass, plastic, or any suitable polymer. In various embodiments, the size distribution of units may be selected to leave space for fluids to flow over the units while in the channel. In various embodiments, there may be no space for fluid to flow over the units. Treatments and chemical reactions described in further detail elsewhere herein may be performed without requiring space for fluid to flow over the units within the channels of the microfluidic devices described herein. For example, treatments comprising the application of heat or light may be performed without such spaces.

[0116] The present invention may include reaction chambers. Various regions within the microfluidic devices described herein, for example branch channels, may be utilized as reaction chambers. Reaction chambers may be enclosed by valves located in or at the end of a channel. Reaction chambers may also be valve-less and the pressure or flow of carrier fluid and / or reagents controlled by pumps with inlets or outlets connecting to the reaction chamber. The units can be flowed from one reaction chamber to another directly or through one or more channel(s). The size of the reaction chamber can vary and may depend on the spacing or size of the valves or pump inlets / outlets defining the reaction chamber(s) and the dimensions, e.g. width, height, diameter, or cross-section of the reaction chamber(s). The size of the reaction chambers can be about, at least, or at least about 10 pl, 20 pl, 30 pl, 40 pl, 50 pl, 60 pl, 70 pl, 80 pl, 90 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 100-200 pl, 200-300 pl, 300-400 pl, 400-500 pl, 500-600 pl, 600-700 pl, 700-800 pl, 800-900 pl, 900-1000 pl, 1 nl, 2 nl, 3 nl, 4 nl, 5 nl, 6 nl, 7 nl, 8 nl, 9 nl, 10 pl nl, 20 nl, 30 nl, 40 nl, 50 nl, 60 nl, 70 nl, 80 nl, 90 nl, 100 nl, 200 nl, 300 nl, 400 nl, 500 nl, 600 nl, 700 nl, 800 nl, 900 nl, 1 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, or more. The size of the reaction chambers can be less than or less than about 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 1000 nl, 900 nl, 800 nl, 700 nl, 600 nl, 500 nl, 400 nl, 300 nl, 200 nl, 100 nl, 90 nl, 80 nl, 70 nl, 60 nl, 50 nl, 40 nl, 30 nl, 20 nl, 10 nl, 9 nl, 8 nl, 7 nl, 6 nl, 5 nl, 4 nl, 3 nl, 2 nl, 1 nl, 900 pl, 800 pl, 700 pl, 600 pl, 500 pl, 400 pl, 300 pl, 200 pl, 100 pl, 90 pl, 80 pl, 70 pl, 60 pl, 50 pl, 40 pl, 30 pl, 20 pl, 10 pl, or less. Those of skill in the art will appreciate that the reaction chambers may have a size that falls within any range bound by any of these values, for example 10-50 nl, 10-100 nl, 50-100 nl, 100-200 nl, 200-300 nl, 300-400 nl, 400-500 nl, 500-600 nl, 600-700 nl, 700-800 nl, 800-900 nl, 900-1000 nl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 1-10 μl, 10-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. Values for the reaction chamber may range between any of the potential values set forth for the reaction chamber herein.

[0117] Channels in which modification procedures occur may have one or more inlet or outlet ports and / or valves. Reagents may be delivered through valve or port into and out of the channel. These inlet or outlet ports and valves may be configured or suitably occluded so as to prevent units from becoming trapped or disarranged. The units may be held in a channel, for example during a modification procedure, by one or more closed, occlusive, or porous valves, gates, switches, or by magnetic fields. Units having permanent or inducible magnetic properties may be employed to utilize their interaction with magnetic fields. A modification procedure may be operated on some or all of the units in a particular channel. In some cases, the selected modification procedure does not cause a change in the unit or the product associated with the unit. Zero or more modification procedures may be applied to units in given channel. Different channels of a fluidic device may be configured to enable distinct modification procedures that can be applied, either sequentially or simultaneously, to the units in the respective channels. Channels may split more than once before converging, separate modification procedures can be applied to any channel.

[0118] In various embodiments, all units intended to receive the application of the same reaction condition(s) are kept in a single channel designated for the application of such reaction condition(s). In some embodiments, units designated to receive the application of the same reaction condition(s) are distributed into a plurality of channels or reaction chambers, including for example branch channels.

[0119] The microfluidic device may contain branch points where the channel splits or divides into multiple channels or outlets. The branch points may comprise about, at least, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more channels or outlets, including without limitation branch channels or reaction chambers. Values for the branch points may range between any of the potential values set forth for the branch points herein. One or more branch points may be arranged sequentially. The branch channels or outlets may have 2-dimensional or 3-dimensional arrangements. For example, a branch point may split a first channel into two or more branch channels in the X, Y plane, resulting in a 2-dimensional planar channel arrangement within the device. Or, a branch point may split a first channel into two or more branch channels in and / or out of the X, Y plane. In such an arrangement, one or more branch channels in a first set may be in one plane A with the portion of the first channel immediately adjacent to the branch point, while the branch-point adjacent portions of one or more branch channels in a second set may be in a different plane than plane A, for example, perpendicular to place A, resulting in a 3-dimensional branch-point channel arrangement within the devices described herein. In some embodiments, one or more channels in the devices described herein are non-linear, for example such devices may have the shape of a spiral, or other curve.Routing of Units

[0120] The microfluidics device described herein can be configured to route units through the device. Routing of units may comprise holding units, moving units, distributing units into channel(s) or branch channel(s) and / or merging units from two or more channels or branch channels to one or more channel(s). The device can also be configured to merge units from two or more channel(s) or branch channel(s) to one or more channel(s). In various embodiments, routing comprises distribution. Units within microfluidic devices described herein may be routed from p locations, e.g. channels, to p+i locations within the microfluidic device, where p, i>0, through a distributor. These p+i locations may be channels generally referred to as branch channels herein. In various embodiments, routing comprises merging. Units within microfluidic devices described herein may be routed from q locations, e.g. channels, into q−j locations, where q, j, q−j>0, through a merger. These q-j locations may be channels generally referred to as merger channels herein. In some embodiments, p is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, p is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, p is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, i is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, i is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, i is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, q is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, q is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, q is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, j is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, j is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, j is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. Values for p, q, j, and / or i, may fall within a range bounded by any of the potential values set forth the p, q, j, and / or i, herein. Routing may comprise the movement of units within a channel, or from one location in a fluidic device to another, or from a first channel to a second channel, where the axis of flow of the first channel may be the same as the second, or alternatively the axis of flow of the first may be at any angle, for example, 45° or 90°, to that of the axis of flow of the second. Distribution may comprise the movement of units from a first channel into a branch channel via a branch point, from one or more branch channel(s) or reaction chamber(s) into one or more other channel(s). Merging may comprise the reverse of distribution. Units may be merged by moving units from q locations within a microfluidic device, e.g. q branch channel(s) or reaction chamber(s) via one or more branch point(s) and into q-j locations within the microfluidic device where q>j, for example into a first channel from which the units had been distributed.

[0121] The microfluidic device described herein can be configured to steer / route units via any appropriate mechanism known in the art, including but not limited to, mechanisms for generating and modulating fluid flow (e.g., as in electroosmotic mechanisms described below), generating or modulating fluidic pressure, moving mechanical mechanisms, static or non-moving mechanical features, or non-moving force generating mechanism. Routers constructed according to such routing mechanisms or any other suitable mechanism known in the art, may be configured and used to move or route units within a first channel, move or route units from a first channel to a second channel, distribute units from a first channel into two or more branch channels, and / or merge units from two or more branch channels into a first or second channel. The microfluidic device described herein may have one, two, or multiple routing mechanisms.

[0122] Fluidic pressure modulation routing mechanisms may include, but are not limited to, mechanisms that increase or decrease the fluidic pressure at one or more locations within a fluidic device. Fluidic pressure modulation mechanisms may comprise any appropriate mechanical device known in the art such as fluidic pumps, gas pressure driven pumps, manual syringes, electronically controlled syringe pumps, electroosmotic pumps, diaphragm pumps, gear pumps, peristaltic pumps, electrohydrodynamic pumps, or any combination thereof. The devices described herein may contain one or more fluidic pressure modulating mechanisms of the same type, or of different types. Fluidic pressure modulating mechanisms may or may not be under specific electronic control, and may have feedback control to ensure appropriate pressure delivery. The fluidic pressure modulating mechanisms may operate independently or be under synchronized control. Not wishing to be bound by theory, units may be moved, flowed, advanced, reversed, held, stopped, directed, and / or redirected in the device by applying increased or decreased relative or absolute pressure to fluids and / or units in the device.

[0123] In one embodiment, routing by the microfluidic device is implemented with inclusion of elements for providing pressure-driven crossflows, for example where a pressure-driven crossflow is configured using fluidic pressures, for example externally generated pressures, which may be gated by one or more valves. The valve(s) associated with the pressure-driven crossflow can be integrated with the microfluidic device (e.g., on-chip), or distinct from the microfluidic device (e.g., or off-chip). In an example, the valve(s) include pneumatic values, for example pneumatic valves actuated at 4 bar at over 300 Hz; however, other pressure levels and frequencies of actuation can be implemented by the microfluidic devices described in further detail herein. Furthermore, the medium used to drive the crossflow can be liquid or gas (e.g. air). Routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000 Hz or more.

[0124] Moving mechanical routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids within a fluidic device. Methods and devices described herein may utilize any suitable moving mechanical routers known in the art, including but not limited to, plugs, pistons, gates, flippers, valves, pins, ratchets, or any combination thereof. Units may be held by a closed mechanical router of a device and / or released upon opening of the mechanical router. Moving mechanical routers may be configured to apply a force either directly to the units, and / or to the fluid in a device described herein such that units may be moved, stopped, held, directed, and / or redirected in the device.

[0125] In one embodiment, routing by the microfluidic device is implemented with inclusion of a mechanical actuator includes a cross-channel fluidically connected to two or more chambers, wherein each of the two or more chambers has at least one compliant side (e.g. the roof) which may be deflected to change the volume of the chamber. In a two-chamber embodiment, a first force applied to the compliant side of the chamber displaces fluid towards the flow channel, and a second force applied to the compliant side of the second chamber displaces fluid from the flow channel into the second chamber. Such a configuration can be connected to two sides of a routing junction of a microfluidic device such one chamber can operate in a “pulling mode” and the other chamber can operate in “pushing mode”, whereby flow can be configured to be purely across the junction, rather than adding fluid flow along the primary flow direction in a manner that opposes or adds to the primary flow. In variations, the embodiment of the mechanical actuator can include elements for providing forces including one or more of: magnetic forces (e.g., with permanent or electromagnets coupled to the compliant side of the chamber); piezeoelectric-provided forces (e.g., with extending and contracting piezoelectric actuators coupled to the compliant side of a chamber, “buckling”-type actuators); mechanical actuators (e.g., mechanical solenoids coupled to and configured to push and pull the compliant side of a chamber); external pressure sources (e.g., gas pressurizers configured to apply and release pressure by one or more pneumatic valves); and other actuators. In some embodiments, the mechanical actuator includes elements for providing forces using surface acoustic waves, transient surface acoustic waves, electrophoresis, dielectrophoresis, micromechanical valves, optical tweezers, and / or thermal vapor bubbles, for example vapor bubbles created by laser absorption or by electrical heating. Routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000 Hz or more.

[0126] In another embodiment, a mechanical component includes a deflector (e.g., flipper valve) that steers flow, beads, or other units toward a desired path or channel. In examples, the deflector(s) can be actuated by one or more of: microelectromechanical systems (MEMS) actuators integrated into a channel network, magnetic actuators providing magnetic fields that actuate magnetic flippers, mechanical actuators / servos, and other actuators for manipulating flippers integrated with the channel network.

[0127] Static or non-moving mechanical routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids within a fluidic device. Such routers may utilize any appropriate static mechanical mechanism known in the art, including but not limited to pillars, grooves, wedges, walls, scallops, holes, cups, divots, sieves, selective stops (e.g. allow fluids to pass, but units are held back), dams, weirs or other similar mechanism, or any combinations thereof. The microfluidic device described herein may comprise one or multiple static or non-moving mechanical routers. The microfluidic device may comprise a single type of static router, for example one or more selective stop, or two or more types of static routers, for example one or more dam and one or more pillar. Such examples are not meant to be limiting. Static or non-moving mechanical routers may be configured to apply a force either directly to the units, or to the fluid in the device such that units may be moved, stopped, held, directed, and / or redirected in the devices described herein.

[0128] Non-moving force generating routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids, including without limitation compositions, such as oligomers within such fluids, within a fluidic device (e.g., thermal vapor bubbles). Such routers may use any appropriate static mechanical mechanism known in the art, including but not limited to electrophoresis, dielectrophoretic, acoustophoresis, electroosmosis, magnetophoresis, gravity, or any combination thereof (see e.g., Wyatt Shields C. et al, Lab Chip 2015 15(5):1230-1249, incorporated herein by reference in its entirety). In one embodiment, the units are routed via magnetophoresis. Non-moving force generating routers may be configured to apply a force directly to the units, and / or apply a force to or through the fluid in the device such that units may be moved, stopped, held, directed, and / or redirected in the devices described herein.

[0129] In one embodiment, a non-moving force generating router includes one or more magnets (e.g., permanent magnets, electromagnets) configured to route units responsive to magnetic fields or magnetic field gradients (e.g., paramagnetic, superparamagnetic beads may be moved by a magnetic field gradient). Magnetic fields and / or field gradients can be generated by devices integrated within or outside the microfluidic devices described herein. Permanent magnets may be actuated toward and / or away from desired regions of the system to affect fields. Electromagnets may be transitioned between on and off states to affect fields. In embodiments, pointed magnets and / or electromagnet cores are used to produce high-field gradients.

[0130] In a related embodiment, microfluidic devices described herein may be configured to implement ferrofluid actuation. A ferrofluid (e.g., a suspension of fine magnetic particles in a liquid medium which is immiscible with the liquid containing beads or other units) is actuated (e.g., by a permanent magnet, by an electromagnet) in response to applied magnetic fields. Such an embodiment can include an operation mode that moves the ferrofluid toward an applied magnetic field in order to displace fluid in a channel, thereby forming a ferrofluid “piston”. Such an embodiment can additionally or alternatively include an operation mode where the ferrofluid is configured to become rigid when subjected to a magnetic field. Upon becoming rigid, the ferrofluid may be used to block one or more of the exits of a routing junction, thereby causing the flow and / or units, e.g. beads, to be routed towards an un-blocked channel.

[0131] In some embodiments, routers described herein comprise or are implemented in cooperation with one or more electroosmotic pumps. Electroosmotic pumps according to the various embodiments described herein, may be controlled electrically in order to generate a flow across a steering junction. For example, an electroosmotic pump can be coupled to or otherwise interface with multiple sides of a steering junction such that it “pulls” on a first side and “pushes” on another side (e.g., in a closed loop). In such a configuration, flow can be configured to be purely or primarily across the junction, as opposed to requiring added fluid flow along a primary flow direction (which would oppose or add to the primary flow). Furthermore, such a configuration can provide fast response speeds. (e.g., providing responses in less than 1 ms).

[0132] In some embodiments, routers described herein comprise or are implemented in cooperation with electrophoretic steering apparatus configured to provide an electric field in solution. For example, units, e.g. coupled to nucleic acid (e.g., DNAs, DNA fragments) can be inherently negatively charged in aqueous solution and manipulated by electrophoretic forces that route such units in a desired manner.

[0133] In some embodiments, routers described herein comprise or are implemented in cooperation with dielectrophoretic steering apparatus configured to provide an electric field in solution. For example, a dielectrophoretic apparatus can be configured to manipulate particles that electrically polarize under the influence of an electric field (e.g., high frequency field on the order of kHz). Without being bounded by theory, if the units' polarization is significantly different than that of the surrounding medium, a net dielectric force is exerted on the particles, which can be used for steering the unit (e.g., at a rate of 400 Hz). Dielectric forces may be applied to units in solution directly; additionally or alternatively. In some embodiments, dielectric forces are applied to beads encapsulated in aqueous droplets in an oil. Dielectrophoretic routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400 Hz or more. In some embodiments, dielectrophoretic routing of units is performed at rates less than 500, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 Hz or less. Dielectrophoretic routers may be configured to route units within the microfluidic devices described herein at a rate that falls within a range bounded by any of the foregoing values, e.g. 100-400 Hz, 10-300 Hz, 20-500 Hz, etc.

[0134] In another embodiment, routers described herein comprise or are implemented in cooperation with charged droplet steering apparatus that encapsulates beads in aqueous droplets in an oil stream. The droplets may be given a net charge. The routers may be used to route the charged droplets, for example steer them at a sorting junction, such as by affecting attraction toward and / or repulsion by charged plates. In various embodiments, routers acting on charged droplets may be configured to route units within microfluidic devices described herein at, at about, or at greater than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 Hz or higher. Routers acting on charged droplets may be configured to route units within the microfluidic devices described herein at rates less than 2000, 1000, 900. 800. 700. 600, 500, 400, 300, 200, 100, 75, 50, 25, 20, 10 Hz or less. Routers acting on charged droplets may be configured to route units within the microfluidic devices described herein at a rate that falls within a range bounded by any of the foregoing values, e.g. 100-1000 Hz, 10-2000 Hz, 20-500 Hz, etc.

[0135] In another embodiment, routers described herein comprise or are implemented in cooperation with an electrostrictive apparatus. For example, electrostrictive apparatus may comprise sources of positive and negative potentials applied to opposing sides of a layer of an elastomeric polymer. Without being bound by theory, such positive and negative potentials may be used to apply forces that cause the polymer to compress. The elastomeric polymer may be coupled to or integrated with a wall of a chamber, such that the applied forces cause the chamber volume to expand and / or contract. In various embodiments, multiple chambers configured to be under the control of electrostrictive apparatus may be coupled to a flow channel via a cross channel (e.g., as described above). A first chamber that may be actuated to pull fluid from the flow channel into the first chamber. If a second chamber fluidically connected to the cross-channel junction is deactivated, fluid may be released from the second chamber to the flow channel.

[0136] In another embodiment, routers described herein comprise or are implemented in cooperation with thermal bubble forming apparatus. Thermal generation of microbubbles can rapidly generate gasses that displace liquid volume. Microbubbles may be used for routing of units, (e.g. beads) in a flow stream, for example, by displacing liquid around a unit and thereby creating a flow stream. In related embodiments, routers comprise or are implemented in cooperation with electrochemical bubble forming apparatus. Electrochemical generation of microbubbles can rapidly generate gasses that displace liquid volume and can be used for routing of units (e.g. beads) in a flow stream.

[0137] Various embodiments of routers described herein comprise microheaters or microactuators configured to generate bubbles. Use of microheaters and microactuators for bubble generation is further described in aus der Wiesche, S. Rembe, C., Maier, C., and Hofer, E. P. Dynamics in Microfluidic Systems with Microheaters (1999) and Chen, C., Wang, J. and Solgaard, O. Micromachined bubble-jet cell sorter with multiple operation modes. Sensors and Actuators B. 2006; 117: 523-529, both of which are herein incorporated by reference in their entirety. See also, Vercruysse, D., Liu, C., Dusa, A. d Wijs, K., Majeed, B. Miyazaki, T. Peeters, S., and Lagae, L. A High-Speed Miniaturized Cell Sorter with Lens-Free Imaging and Thermal Bubble Based Jet Flow Sorting. 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2014: 382-384; UK Patent Application No. GB 2561587; and European Patent Application No. EP2602608, all of which are herein incorporated by reference in their entirety. Without being bound by theory, bubbles generated within a fluid of substantially laminar flow may cause a displacement of the fluid and / or unit(s) within the fluid laterally with respect to the direction of flow, e.g. by a force that disturbs the fluid and / or unit(s) within the fluid laterally with respect to the direction of flow.

[0138] In some embodiments, routers described herein comprise a vortex element or elements configured to generate a vortex in a flow stream within a desired distance from the microactuator, e.g., within about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm or more from the microactuator. Without being bound by theory, a vortex generated downstream from a microactuator configured to generate bubbles in the fluid moving along a channel may be used to amplify the displacement of the fluid and / or unit(s) within the fluid and / or the force that disturbs the fluid and / or unit(s) within the fluid. For example, a displacement and / or force generated by a microactuator configured to generate bubbles upstream from the vortex element may be amplified by the vortex. In some cases, the vortex elements configured to generate a vortex may comprise a recess, turn, and / or protrusion in a channel within a microfluidic device (e.g., a triangular recess on the channel wall). The generated vortex may travel downstream with the unit to be sorted and may cause routing of the unit into a branch channel. In some embodiments the vortex generating element is located between a microheater or microactuator configured to generate bubbles, and a branch channel.

[0139] In some embodiments, microfluidic devices described herein comprise a microactuator configured to generate bubbles, one or more vortex elements configured to generate a vortex and / or a branch point configured to route a unit in flow into one of a plurality of branch channels, located within 100 μm to 10 mm along the direction of flow. The path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may have a length of at least 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 1000 μm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, or more. The path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may have a length of at most 10.0 mm, 9.0 mm, 8.0 mm, 7.0 mm, 6.0 mm, 5.0 mm, 4.5 mm, 4.0 mm, 3.5 mm, 3.0 mm, 2.9 mm, 2.8 mm, 2.7 mm, 2.6 mm, 2.5 mm, 2.4 mm, 2.3 mm, 2.2 mm, 2.1 mm, 2.0 mm, 1.9 mm, 1.8 mm, 1.7 mm, 1.6 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.0 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, or less. The length of the path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may fall within a range bound by any of the foregoing values, e.g. 1.6-8.0 mm, 750 μm-2.3 mm, or 100 μm-3.5 mm.

[0140] In some embodiments, microfluidic devices comprising routers comprising or implemented in conjunction with a thermal bubble forming apparatus comprise one unit delivery channel and one or more (e.g. two) fluid delivery channels that all open into a switch chamber with two branch outlet channels. The fluid delivery channels may be connected to a common fluid source. In some embodiments, the fluid delivery channels terminate in a firing chamber and a nozzle connected to the switch chamber. In some embodiments, one or more microactuator(s) (e.g. microheater(s)) is incorporated into the wall of the firing chamber. The microactuator may be used to generate a bubble, e.g. following application of an electrical pulse. Growth of the bubble may force the incoming fluid from a fluid delivery channel to pass through the nozzle and form a jet that pushes a selected unit toward the desired (e.g. the farther) branch outlet channel. Timing of the application of the electrical pulse may be calibrated to cause incoming units to enter into a designated branch outlet channel.

[0141] The routers comprising or implemented in cooperation with a thermal bubble forming apparatus described herein may be fabricated using any suitable method known in the art, including without limitation photolithography and deep reactive ion etching (DRIE) to form microfluidic channels and etch on an opposite side of a silicon wafer housing the microfluidic channels openings providing connections to the channels and access to actuator pads. Optical access may be provided by any suitable material known in the art, such as by Pyrex wafers covering the microfluidic channels. Bubble actuator circuits may be patterned on a suitable medium known in the art, e.g, Pyrex. In one example, trenches about 1 μm deep are etched on Pyrex wafers using hydrofluoric acid (HF); a layer of about 400 Å titanium is sputtered on the Pyrex and patterned by a lift-off process to construct microheaters; conducting wires are made by successive sputtering and lift-off of 3000 Å gold; an insulating layer of 3000 Å silicon nitride is deposited on Pyrex by plasma-enhanced chemical vapor deposition (PECVD) and patterned by plasma etch; and Pyrex wafers are anodically bonded to Si wafers. Heaters, conductors and / or insulators may be patterned in the Pyrex trenches. The construction of routers comprising or implemented in cooperation with a thermal bubble forming apparatus (e.g., bubble-jet distributors) are described in Chen, C., Wang, J. and Solgaard, O. Micromachined bubble-jet cell sorter with multiple operation modes. Sensors and Actuators B. 2006; 117: 523-529, which is incorporated herein by reference in its entirety.

[0142] In another embodiment, routers described herein comprise or are implemented in cooperation with acoustic steering apparatus. An acoustic steering apparatus may be configured to generate sound / pressure waves and apply such waves to flow within the system (e.g. in a way that produces standing waves of low and / or high pressure). Standing acoustic waves may be used to route units, e.g. beads in the microfluidic devices described herein. In some embodiments, transient surface acoustic waves are used to route units.

[0143] In another embodiment, routers described herein comprise or are implemented in cooperation with electrowetting apparatus that generates electric fields / electric charges. Electric fields and / or charges may be configured to modulate surface tension at corresponding liquid-gas, liquid-solid, and / or liquid-liquid interfaces. Modulation of surface tension may be used to maintain or release pressure gradients that cause fluids to remain still or move under resulting modified pressures provided by the electrowetting apparatus, thereby routing units (e.g. beads in fluid communication with the modulated fluids).

[0144] Routers as described further herein may be configured to merge one or more units from different channels or branch channels. For example, a router may be configured to merge one or more units from a first channel to a second channel, from two or more channels into a single channel, or from two or more branch channels to a second channel. A single type of router or any combination of routers may be used in a single device. The sequencing of moving specific individual units or sets of units into to specific locations within a device, or from one channel to another, or from two or more branch channels into a single destination channel may be controlled by a single type of router or a combination of different types of routers as described in further detail herein. The movement of units into one or more channels may be verified by one or more detectors.

[0145] The microfluidic device described herein may be configured to distribute one or more units from one channel to one or more channels or branch channels via any appropriate distribution mechanism known in the art. The devices described herein may comprise one or more types of distributors. Distributors in a microfluidic device can be configured to stop, hold, direct, or redirect units or fluid in the device. A distributor may be used to close off sections of the channel(s) or branch channels, or to impede progress of the units through or into a channel or branch channel.

[0146] Distributors in a microfluidic device may be configured to distribute one or more units from a primary channel into one or more branch channel(s) based on the positional order of the unit(s) in the primary channel. Distributors may also be configured to distribute one or more units into one or more branch channel(s) based on a label on a unit. The distribution of one or more units to a branch channel may be predesignated according to an intended sequence of reactions and / or treatments. The intended sequence of reactions and / or treatments may be preassigned to the one or more unit. The channels to which the one or more units may be distributed may also be randomly assigned to the one or more unit. Methods to distribute one or more units into a channel or branch channel include, but are not limited to, altering the position of a unit within the laminar or laminar-like flow at or before a branch point; the presence of one or more moving or non-moving mechanical devices at or before a branch point to direct units into a channel or branch channel; any method that alters the amount or pressure of the fluid flow through branch channels such that units are directed into one or more branch channel(s), or any combination thereof or any other suitable method known in the art. The correct distribution of one or more units into one or more branch channel(s) may be verified using detectors. Incorrectly distributed units may be subjected to an error correction mechanism described elsewhere herein, for example, by directing and / or holding one or more units into a side channel and / or redirecting the one or more units in a side channel back into a primary channel holding positionally ordered units, and / or any other suitable error correction mechanism known in the art.

[0147] In some embodiments, the units are distributed by altering the position of the unit within the fluid. Such methods can alter the position of the unit within ordered flow, for example within laminar or laminar-like flow, of a channel. Lateral movement of a unit within flow may cause the unit to be directed into a desired channel at a branch point, typically the channel located at the same side as the unit's relative position within flow prior to the branch point. Methods that alter the position of a unit within the flow include the application of electrostatic or electrokinetic forces such as electrophoresis, dielectrophoresis, and electroosmotic flow; acoustic forces such as bulk standing waves, standing surface acoustics waves, and traveling waves; optical manipulation(s) or optical radiation with focused laser beam(s), also known as optical tweezers; application of side flow or cross flow at an angle to the flow direction of a unit to move the unit laterally within the flow; gravity; magnetophoresis if the units contain ferromagnetic materials; flow focusing; via the application of any other suitable type of force known in the art; or combinations thereof. In some embodiments, application of side flow or cross flow is performed by application of pressure, electroosmosis, or displacement via pistons or actuators, such as those comprising piezoelectric, electrostatic or electroactive polymers, or pumps such as electoosmotic pumps.

[0148] In some embodiments, the units are distributed by moving mechanical distributors. Moving mechanical distributors that may be configured to distribute units include, but are not limited to, rotary valves, ratchet mechanisms, pins, flippers, gates, flow switching mechanisms, or channel actuation via application of heat to a thermoreversible gelation polymer.

[0149] In some embodiments, the units are distributed by methods that alter the fluidic pressure of a channel, including without limitation a branch channel. This method can be used to cause increased or decreased fluid from one channel to flow into another specified channel at the branch point. For example, as the relative pressure is increased in one channel and decreased in a second, connecting channel, the carrier fluid and units carried therein can be directed into the second channel with the lower relative pressure.

[0150] Routers, e.g. distributors, having suitable configurations as described in further detail herein may also be used as mergers to merge units from at least q channels into q-j channel(s), where q>j. For example, units from two channels may be merged into one merger channel, or units from four channels may be merged into three, two, or one merger channels. Differential pressures may be utilized to cause release of units from two or more branch channels into one or more channels in a designated order. By application of a lower relative pressure into a first branch channel, units therein may be kept from entering the branch point and / or an adjacent merger channel while units from a second branch channel leading to the same branch point may be released from the second branch channel into and / or past the branch point. Such units may be routed into the merger channel prior to the release of units from the first branch channel into the branch point and / or merger channel.

[0151] In various embodiments, dedicated routers, e.g. distributors, are used to facilitate the movement and / or merging of mobile units. For example, a router, e.g. a distributor, placed at the branch point of two channels can be configured to direct one or more unit into one or more channel(s) or branch channel(s) during distribution. In the reverse direction, the same router may block, hold, or impede the movement of units from a first branch channel while allowing the movement of units from a second branch channel into a single channel, thus allowing the controlled and / or orderly distribution of units as well as the controlled and / or orderly merging of units. Distribution of units into branch channels may comprise distributors that act on one or more units with spacing between them. Units may be merged from p channels into p-b channels, where p>b, via use of any router, e.g distributor, to route one or more units in a first channel and then route one or more units from a second channel.

[0152] In various embodiment, microfluidic devices and systems comprise one or more of the following: high-speed routers, e.g. distributor(s), for directing units into one of the multiple branch channels, e.g. for parallel synthesis; high-speed unit counting sensor(s) configured to detect units prior to a distribution step; and device integration that combines discrete components, for example unit router(s), unit detector(s), multiple capillaries, and / or reagent mixing chips into a complete device.

[0153] The position of the units in the device may be maintained by a variety of methods. For example, the position of the units in the device may be maintained by placing physical constraints on the units in a channel(s) to preserve the relative position of the units or by spacing the units in a channel(s) under continuous flow or by a combination of both within the same device. To place physical constraints on units, a channel width may be selected to be sufficiently narrow so that units cannot pass one another in the channel. To maintain order of units in flow, e.g. in laminar or laminar-like flow, the units may be separated in continuous or stopped flow with sufficient space between the units that they do not pass one another during the flow or during stopped flow. While uncontrolled migration of the units due to factors like, but not limited to, diffusion, or sedimentation may eventually cause units to pass one another, stopped flow for short periods of time can maintain order of sufficiently spaced units for desired periods of time.

[0154] The microfluidic device described herein may also correct unit position errors introduced during the operation of a microfluidic device described herein, for example during operation for nucleic acid synthesis. Additional routers and channels may be added to the system to handle units that have been incorrectly distributed. Units incorrectly distributed at a first router may be routed into a second channel where correct distribution can be performed immediately. For example, a channel comprising a loop can return a unit to a position before the distribution router such that the unit can be correctly routed. Units can also be routed into branch channels and held for either the remainder of device operation, or they can be held temporarily and subsequently routed back to into position to be distributed.

[0155] In some cases, two or more neighboring units may exchange position while not affecting other units on either side of the exchanged units. In various embodiments, such units getting out of positional order are identified by a detector. This type of error may result in incorrect reactions, treatments, or modifications being applied to the units, e.g. incorrectly synthesized molecules on affected units. In some embodiments, this error occurs at less than 0.000001 times, 0.00001 times, 0.0001 times, 0.001 times, 0.0025 times, 0.005 times, 0.0075 times, 0.01 times, 0.025 times, 0.05 times, 0.075 times, 0.1 times, 0.25 times, 0.5 times, 0.75 times, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 30 times per unit per 100 cycles of modification.

[0156] In some cases, one or more units may be incorrectly distributed through mis-routing at a branch point. In various embodiments, mis-routed units can be identified by a detector. In some embodiments mis-routing can be detected in the channel in which reactions or treatments occur. In some embodiments, mis-routing can be detected after mis-routing by detectors placed after the branch point. In some embodiments detection of the mis-routing event can occur at any point between the branch point and the channel in which the reactions or treatments occur. The effect of this type of an error may be limited to only the mis-routed units. Subsequent units may be correctly routed, and only the mis-routed unit may be affected by the mis-routing. In some embodiments, the mis-routing is detected, and the positions of all units is updated accordingly so that the history of each unit is known and units with the desired sequence of treatments can be identified and / or from those without the desired sequence of treatments, e.g. nucleic acid synthesis steps.

[0157] In various embodiments, additional routers and channels may be added to the microfluidic device system to hold units that have been incorrectly distributed. In some embodiments, a mis-routed unit may be detected and routed into a branch channel for holding until the unit can be routed back, for example for further distribution. In some embodiments, treatments and chemical reactions are reserved from mis-routed units held in such channels. Units can also be routed into branch channels and held for either the remainder of device operation or discarded. Units incorrectly distributed at a first router, e.g. a distributor, may be re-routed into a second channel where correct distribution can be attempted immediately, such as a channel comprising a loop that returns a unit to a position before the distributor such that another attempt at correctly routing the unit can be made. In various embodiments, the positional information of the mis-routed units and all other units is updated, such that the position and history of all or a subset of the units throughout the device remains known. In some embodiments, these types of errors may be tracked or corrected such that they do not result in a loss of correct sequence of treatments or modifications applied to or to be applied to some or all units.

[0158] In some embodiments, this type of a mis-routing error occurs at, less than or less than about 0.000001 times, 0.00001 times, 0.0001 times, 0.001 times, 0.0025 times, 0.005 times, 0.0075 times, 0.01 times, 0.025 times, 0.05 times, 0.075 times, 0.1 times, 0.25 times, 0.5 times, 0.75 times, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 30 time per unit per 100 cycles of modification. Values for the error rates may range between any of the potential values set forth for the error rates herein. In some embodiments, mis-routed units may escape detection. This type of error may result in incorrect synthesis history for units that are out of positional order. In some embodiments, labeled units capable of labeling units, for example beads that can be colored with pigment or imbued with fluorescent properties, are used to verify routing. Detectors at any point in the device or on any cycle of operation may be used to verify such labeled units are in the expected relative position. In one embodiment for example, one in 100 beads in a device may be labeled with a fluorescent dye. During device operation, the relative positional ordering of these labeled and distinguishable beads may be verified against their expected position based on predesignated routing paths for each of the units. In some embodiments, the verification occurs in reaction channels after each cycle of device operation. In other embodiments verification occurs on each cycle in the initial channel prior to distribution. In further embodiments verification occurs only once after all cycles are complete and all modifications have taken place.

[0159] In various embodiments, devices and systems described herein are operated for multiple cycles, where all or substantially all of the units within a microfluidic device are returned to a common area, such as a channel. Unit detection, identification of mis-routing events, corrective routing may be performed one or more times during each cycle of operation.Unit Spacing

[0160] In various embodiments, units are held and moved together in a group having units adjacent to each other in a channel. This “stacked regime” may comprise units that are held or flowed in direct contact with (e.g. end-to-end and / or with their geometric centers offset) or in close vicinity of each other. In various embodiments, the order of units within a channel is maintained by the restrictive width of the channel holding the units, preventing units from swapping positions outside of their order. The ratio of unit diameter and / or sizes to channel diameter, cross-section, or widths can be selected to maintain positional ordering and / or to prevent wedging of units within a channel which may lead to clogging.

[0161] Without wishing to be bound by theory, units moving through a microfluidic device in a stacked regime can contact each other and the channel at acute angles, creating a force that may push the units into the channel walls. This may result in the likelihood of the units wedging and clogging the channel. Such forces may become so great as to distort or compress the units such that the units stop moving in the channel. In addition, imperfection in the unit surface may likewise prevent movement through the channel. Without wishing to be bound by theory, a solution to units clogging in the stacked regime includes the use of straight and sufficiently smooth channels, and / or units that are sufficiently smooth and / or round. Channels that are straight and sufficiently smooth can support movement of beads in the stacked regime. In addition, unit spacers may be incorporated into the microfluidic devices described herein to separate stacked beads in channels with changing dimensions, e.g. at width transitions, or at branch points.

[0162] Units in the stacked regime may be abutting or touching one another in the channel(s). In some embodiments, units are less than 1 unit length apart in the direction of the flow, e.g. due to the offset geometric centers within a channel. Units may be a fraction of a unit length apart. In some embodiments, units are about, less than, or less than about 2, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05 or fewer unit lengths apart, center to center, in the direction of the flow. Center to center unit spacing in the direction of the flow may fall within any range bound by the foregoing values, including for example 0.1-0.2, 0.1-1, 0.2-0.3, 0.2-1.5, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, or 0.9-2 unit lengths. Values for the unit spacing may range between any of the potential values set forth for the unit spacing herein.

[0163] In various embodiments, units are separated by space from each other. This “separated regime” may facilitate proper distribution by allowing various routers, e.g. distributors, to act on units individually, without interference from other units; may allow units to navigate various features or aspects of the device that could briefly or temporarily slow or impede the movement of a unit such as a corner, constriction, edge, expansion, or combination thereof without risk of clogging due to interference or contact by adjacent units; and may allow units to move to and from areas of the device in ordered flow, e.g. in laminar or laminar-like flow. Flow-based unit ordering, e.g. in laminar or laminar-like flow can be used to allow the use of channels that are greater in width than those allowed in a stacked regime. Ordered flow may be maintained in separated regime in channels having greater widths than widths that allow for maintaining unit order by physically constraining unit mixing, including without limitations widths that are about, more than, or more than about 2 times the width of unit size.

[0164] In a flow-based unit ordering regime, units may be maintained within channels having widths that are about, more than, or more than about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times the average or nominal diameter and / or size of the units. Values for the channel widths may range between any of the potential values set forth for the channel widths herein.

[0165] Also provided herein are methods for separating units. A spacer or ejector device may be configured to apply a fluidic shear force that results in a first unit accelerating away from a second unit as the first unit passes through the spacer or ejector device. The acceleration of the first unit may introduce space and / or additional fluid volume between the first and second unit.

[0166] Units may be moved through a channel feeding into a unit spacer in various configurations, including, without limitation, individually or as a stacked column. When a unit reaches a unit spacer, e.g. at a T-intersection, an in-line spacer channel, or any suitable cross-channel geometry, units may be separated by the additional flow, or “cross flow,” in the main channel. The additional flow(s) for spacing fluids (e.g., for a T-intersection, for a cross-channel, for an inline-spacer channel for other geometries, etc.) entering from side channels may be created without additional pressure sources. For instance, a bypass channel with low fluidic resistance may be used to redirect fluid upstream of a stacked unit (e.g. bead) column and into the spacer. The lower fluidic resistance may be configured to yield a higher flow rate through the bypass channel than along the unit stack. Increased flow rates may be used to induce high shear and a degree of unit separation. Unit separation may be influenced by channel geometries near the spacer.

[0167] Additionally or alternatively, in associated embodiments, the systems described herein comprise one or more spacers (e.g., spacers having independent pressure control a bypass channel) fluidly interfaced with the channels of a microfluidic device. Such spacers may be used to restack beads or other units in a controlled manner. In operation, such a spacer may be used to reduce the spatial extent of a collection of beads or other units in a portion of the system. In various embodiments, such spacers are configured in conjunction with or without unit (e.g. bead) stops. In more detail, the system can allow fluid flow, with beads or other units, through such spacers in reverse, such that one or more beads can be returned in reverse manner through the spacer to allow restacking or re-ordering of beads in a controlled manner. Additionally or alternatively, such spacers may be used with a bypass channel and an element (e.g., valve, check-valve, variable flow restriction unit, one-way flow restriction unit, etc.) that prevents return flow through the bypass channel, such that beads or other units can be removed from a stack, and not re-stacked. Any of the above spacer embodiments can include wall geometries designed to ensure that beads enter the exit channel without jamming. In one example of a wall geometry that mitigates jamming, the wall geometry can include a narrow gap or slit in the channel wall (e.g., primary channel wall, cross-flow channel wall, etc.). Such a gap or slit may have a width that is less than the diameter of one unit, e.g. bead or other characteristic dimension of a bead or other unit. In examples, the gap or slit width can be less than 20 nm, 100 nm, 500 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, etc. Those of skill in the art will appreciate that the slit or gap width may have a value that is within any range bound by any of these values, for example 20-100 nm, 100-500 nm, 500-1000 nm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, etc., with associated tolerances as described above.

[0168] In one example, units entering a unit spacer having a cross-channel geometry from a feeding channel may enter into a cross-flow incoming laterally to the unit's flow. FIG. 22D provides an illustrative implementation of a unit spacer with cross-flow geometry constructed in accordance with the embodiments herein. The cross-flow may be generated by flow from opposing or substantially opposing directions. The cross-flow may be perpendicular or substantially perpendicular or have a component of velocity perpendicular or substantially perpendicular to the units' path through the unit spacer. The cross-flow may be provided by two or more channels leading into the cross-channel geometry of a unit spacer. A first unit may flow past the cross-channel geometry, followed by a mix of fluid from each side of the cross-flow. In some embodiments, the pressures in channels generating the cross-flow are adjusted such that they are equal and greater than the pressure in the downstream portion of the entering unit's path and less than the pressure in the feeding channel. The pressures in channels generating the cross-flow need not be equal. Unequal flows may be used according to various embodiments, for example to bias flowing units laterally with respect to the units' direction of flow. Suitable pressures, pressure differentials, and / or flow rates, flow rate differentials for causing a desired movement of a unit within the microfluidic devices described herein may be selected as described in further detail elsewhere herein or as is otherwise known in the art.

[0169] A spacing may be generated between the first unit and a second unit entering the unit spacer subsequent to the first unit by the mix of fluid from each side of the cross-flow being introduced between the first and the second channel as they move past the unit spacer. In some embodiments, e.g. for a T-intersection type unit spacer, the cross-flow is provided by one channel. The spacing introduced between units may be used to facilitate subsequent distribution of each unit at a branch channel, various embodiments of which are described in further detail elsewhere herein, by allowing that the router, e.g. a distributor, act on units individually for each distribution event. Therefore, entry of a plurality of units into a router at once may be prevented by introducing a space between units moving in channels of the devices described herein.

[0170] Units may also be spaced from each other in the channel. The units may be spaced by a spacer length of about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 50,000, 100,000 or more unit diameter and / or sizes apart. The units may be spaced by a spacer length of about, less than, or less than about 100,000, 50,000, 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less unit diameter and / or size apart. The spacer length between units may fall within any range bounded by the foregoing limits, including without limitation, between 1-10, 20, 20-30, 30-50, 50-100, 100-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-7500, 7500-10,000, 10,000-50,000, 50,000-100,000 unit diameter and / or sizes. The units may be spaced by a spacer length of about, more than, or more than about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 14 μm, 15 μm, 16 μm, 18 μm, 20 μm, 25 μm, 50 μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, 10,000 μm, 50,000 μm, 100,000 μm or more. The units may be spaced by a spacer length of about, less than, or less than about 100,000 μm, 50,000 μm, 10,000 μm, 9000 μm, 8000 μm, 7000 μm, 6000 μm, 5000 μm, 4000 μm, 3000 μm, 2000 μm, 1000 μm, 900 μm, 800 μm, 700 μm 600 μm, 500 m, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm or less. The spacer length between units may fall within any range bounded by the foregoing limits, including without limitation between 0-10 μm, 20 μm, 20-30 μm, 30-50 μm, 50-100 μm, 100-250 μm, 250-500 μm, 500-1000 μm, 1000-2500 μm, 2500-5000 μm, 5000-7500 μm, 7500-10,000 μm, 10,000-50,000 μm, 50,000-100,000 μm. Values for the unit spacing may range between any of the potential values set forth for the unit spacing herein.Pressure Differentials

[0171] In various embodiments, units flowing through the channels and branch points of the microfluidic devices described herein, may be routed in a designated direction by adjusting pressures and / or flow rates within channels that connect through branch points. FIG. 31 shows exemplary pressure settings in channels connected through one branch point. Without being bound by theory, fluid within microfluidic devices flows down a gradient in pressure. Further, in various channel configurations, pressure drops continuously along the direction of flow. Further without being bound by theory, flow rates through channels of microfluidic devices correlate with the pressure differential between two points (Ptop−Pbottom) divided by the channel length between such two points (FIG. 31A).

[0172] In a branch point of a main channel intersecting with a branch channel (FIG. 31B-F), pressures at locations a distance away from the branch point may be adjusted to set a pressure value at the branch point P0. In FIG. 31B, pressures are adjusted such that Ptop>Pbottom in the main channel and the pressure value at the branch point P0 is equal to Pbranch (P0=Pbranch). In FIG. 31C, pressures at the corresponding locations are adjusted such that Ptop>Pbottom and P0>Pbranch, leading to flow from top to bottom of the main channel as well as from branch point into the branch channel. In FIG. 31D, pressures at the corresponding locations are adjusted such that Ptop>P0>Pbranch and Pbottom>P0>Pbranch, leading to flow from the top and the bottom of the main channel into the branch channel from the branch point. In FIG. 31E, pressures at the corresponding locations are adjusted such that Ptop>Pbranch>P0>Pbottom, leading to flow from the top of the main channel as well as the branch channel toward the bottom of the channel. In FIG. 31F, pressures at the corresponding locations are adjusted such that Pbranch>P0>Ptop and Pbranch>P0>Pbottom, leading to flow from the branch channel to the top as well as the bottom of the main channel. Pressure differentials can be created by setting pressures in a variety of location within microfluidic devices described herein to route flow and / or units carried therein in designated directions following a pressure gradient.

[0173] FIG. 32 provides further exemplary embodiments using pressure differentials to route units within microfluidic devices described herein. A branch point configuration with a main channel and two branch channels B1 and B2 is illustrated describing pressure values at the top and the bottom of the main channel Ptop, Pbottom, respectively, at the intersection of the main channel with the first branch channel B1 and B2, P1, P2, respectively, and at the distal ends of branch channels B1 and B2, PB1, PB2, respectively. FIG. 32B-E provide exemplary values for each of these pressures and resulting flow patterns. For example, flow between the intersection of the main channel with branch channel B1 and the intersection of the main channel with branch channel B2 is governed by the pressure differential P1−P2. Where P1−P2=0, there is no flow between these points (FIG. 32B). Similarly, flow into and out of the first branch channel B1 is governed by the pressure differential P1−PB1; flow into and out of the second branch channel B2 is governed by the pressure differential P2−PB2; and flow between the top of the main channel and the intersection of the main channel with branch channel B1 is governed by the pressure differential Ptop−P1; and flow between the bottom of the main channel and the intersection of the main channel with branch channel B2 is governed by the pressure differential Pbottom−P2.

[0174] Using the pressure differentials exemplified with the pressure values shown in FIG. 32B-E, units may be selectively loaded into branch channel B1 (FIG. 32B) or into branch channel B2 (FIG. 32C). Similarly units can be unloaded selectively from either branch channel. FIG. 32D shows a pressure differential setting for unloading selectively from branch channel B2 toward the top of the main channel. Similar pressure values are set in FIG. 32E as FIG. 32D, except that Pbottom>P2 in FIG. 32E, allowing for flow from the bottom of the main channel past the intersection of the main channel with branch channel B2. Thus, as units are unloaded from branch channel B2 toward the top of the main channel, fluid flowing from the bottom of the main channel is introduced between units creating spacer lengths of fluid (FIG. 32E). In contrast, Pbottom=P2 in FIG. 32D, resulting to no flow from the bottom of the main channel toward the intersection of the main channel with the second channel B2. Such a setting allows the spacing between units to be maintained as the units enter the main channel from the branch channel (FIG. 32D).

[0175] Those skilled in the art will note that similar applications of pressure differentials between various points in microfluidic channels can be used to route, including without limitation to hold, units within microfluidic devices described herein and / or adjust spacing between units.Multi-Branch Channels

[0176] In various embodiments, the microfluidic devices described in further detail herein, comprise routers and branch points with multiple branch channels. In some embodiments, such branch points comprise multiple “sub-branch points” allowing for units to be sorted into branch channels that branch off in consecutive sub-branch points. FIG. 39 depicts an exemplary illustration of a router configured to distribute beads into a plurality of branch channels and / or merge beads from a plurality of branch channels. Incoming units, e.g beads 3901 as depicted in FIG. 39, as they move from the unit spacer 3902 toward the first router 3911, may be routed into a branch channel 3904, via one of the routers 3911, 3912, 3913, 3914. The units may be stopped with a unit stop (e.g. a bead stop) 3921, 3922, 3923, 3924, within and / or at the end of the branch channel. The units may be merged back into the main flow, for example through a spacer 3903, and / or a router 3911, 3912, 3913, 3914. As the units are distributed from and / or merged into the main flow in the main capillary 3910, fluids may be flowed past the branch point, e.g. into a waste reservoir.Units

[0177] The units may be solid or porous. They may or may not carry an attached library product. The units may be glass, polymeric beads, droplets, or cells. The units may be directly modified by the modification procedures described herein. In some embodiments, both a unit and an associated product is modified by one or more modification procedures described herein. Large collections of units can be generated with specific properties such as color, surface chemistries, labels using the various modification procedures described herein. Some or all of the units within a microfluidic device or a channel thereof may be uniquely encoded, without redundancy. The units may be randomly assigned or assigned based on some physical, chemical, or optical characteristic of each unit. A series of modification procedures may be applied sequentially, in a loop or in series, such that each unit is exposed to a particular set of modification procedures. The positional encoding according to the various embodiments of the invention allows the elimination of redundancy. Accordingly, large numbers of physically encoded library units may be generated at low cost. Such library units may be encoded uniquely. Physically encoded library units may be used in downstream procedures. A first procedure where units are physically encoded may be coupled with a second procedure where products are generated on the units, while preserving the positional encoding between the first and second procedures. This approach can be used to associate physical unit encodings with products. By associating the physical encodings with products, the units can be directed into unrelated procedures where the positional information / encoding may be lost, but physical encoding can be detected.

[0178] The units used in various embodiments can be made from a range of materials. In some embodiments, the units are solid. In some embodiments, the units are porous. In some embodiments, the units do not carry an attached library product. The units may be glass, polymeric beads, droplets, bubbles, slugs, or cells. Materials used for beads can include polymers such as polystyrene, melamine resin, polyacrylonitrile, or agarose; hydrogels such as alginate or chitosan; silica, glass, or controlled porous glass (CPG); and metals such as gold, silver, GaAs, GaP, or iron. Silica may be fused silica (amorphous pure silica), quartz (crystalline pure silica), or other generic glass (silica crystalline or amorphous). Many beads can be purchased from vendors such as ThermoFisher or Sigma Aldrich with or without pre-functionalized coatings, including functionalized coatings with reactive chemistries, affinity tags such as biotin or streptavidin, and / or dyes, such as fluorescent dyes. Units may already have a molecule, for example a nucleic acid on their surface while a second, distinct, chemical or molecular compound is added to their surface, or to such molecule during device operation.

[0179] In some embodiments, the units comprise superparamagnetic beads. Without being bound by theory, use of superparamagnetic beads may reduce the number of fluidic connections. In some embodiments, superparamagnetic beads have a high magnetic susceptibility, and are mechanically sound, chemically functionalized, and / or monodisperse.Barcodes

[0180] Units may be barcoded with physical properties, molecular properties, color or pigment, metal, or spectral properties, or any combination thereof. Physical properties include, but are not limited to, etching or shape, or metal bars or deposits. Molecular properties include, but are not limited to, chemical functionalization and chemical compounds, nucleic acids, or biotin or streptavidin affinity tags. Color or pigments include, but are not limited to, fluorescent or non-fluorescent dyes. Barcodes could be used before, after, or during to establish the identity of units prior to commencing operation, during operation to verify the identity of units, or after completion of operation to enable tracking of units after removal from the device and disordering. The identify of barcoded beads may detected and mapped to a unit position so that barcodes need only be read once while positional information is used during operation. Barcodes may be detected at the end of operation to verify correct position.

[0181] Units with or without barcodes or labels may be randomly arranged initially. Units may also be arranged in a known pattern, either due to a deliberate arrangement initially, or as a result of a previous round of synthesis performed using positional encoding. In various embodiments, barcodes (labels) comprise nucleic acids. Barcodes may differ in a variety of chemical or physical feature, such as optical or spectral features, including features described herein or any suitable features known in the art. In some embodiments, barcode differ in more than one type of a feature. For example, a barcode having an optical feature may also comprise an oligonucleotide feature. Multiple features of a barcode can be used in combination to identify one or more features about a unit and / or molecules associated with a unit. For example, one feature of a barcode may be used for identification of a unit. Another feature of a barcode may be used for identification of a target associated with the unit.

[0182] In various embodiments, barcodes are known nucleic acid sequences that allow some feature of a polynucleotide with which the barcode is associated to be identified. In some embodiments, a barcode comprises a nucleic acid sequence that when associated with a target polynucleotide serves as an identifier of the sample from which the target polynucleotide was derived.

[0183] Nucleic acid barcodes can be designed at suitable lengths to allow sufficient degree of identification, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or more nucleotides in length. Multiple barcodes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more barcodes, may be used on the same molecule, optionally separated by non-barcode sequences. In some embodiments, barcodes are shorter than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. In some embodiments, barcodes associated with some polynucleotides are of different length than barcodes associated with other polynucleotides. In general, barcodes are of sufficient length and comprise sequences that are sufficiently different to allow the identification of samples based on barcodes with which they are associated. In some embodiments, each barcode within a plurality of barcodes differ from every other barcode in the plurality at at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more positions.

[0184] In some embodiments, units described herein, such as all or some of the units within a microfluidic device described herein, are associated with one or more of a) a unit-specific barcode uniquely identifying the unit, b) a target specific barcode, and c) a target, e.g. an oligonucleotide. Unit-specific barcodes may be associated with the unit alone or with the unit and the target-specific barcode and / or target. For example, a unit-specific barcode may be attached to a unit and a target-specific barcode and the target-specific barcode may be attached to the target. In some embodiments, unit-specific barcodes are associated with the unit, but are not attached to the target-specific barcode and / or the target. In some embodiments, unit-specific barcodes and target-specific barcodes are linked, e.g. covalently. Target-specific barcodes may be associated with targets in a variety of ways, for example covalently. In some embodiments, a first end of a unit-specific barcode is attached to a unit, the second end of the unit-specific barcode is attached to the first end of a target-specific barcode and the second end of the target-specific barcode is attached to a target. In some embodiments, unit-specific barcodes are attached directly to targets associated with the unit. Target-specific barcodes may also be attached to the target, e.g. through the opposite end of the target from the unit-specific barcode. In some embodiments, unit-associated target-specific barcodes continue to be associated with and / or identify targets after being separated from a unit. In various embodiments, target-specific barcodes for each of the targets on a unit are different. In various embodiments, the unit-specific barcode for each of the targets associated with a unit is the same. Unit-specific barcodes and target-specific barcodes can facilitate methods of target analysis, such as analysis comprising counting. Such analysis may comprise sequencing, hybridization and / or any suitable method known in the art. Such analysis may follow an amplification step. For example, upon amplification of the target along with one or more barcodes described herein, the number of different target-specific barcodes can indicate the number of pre-amplification target molecules. In various embodiments, upon amplification of the target along with one or more barcodes described herein, the number of different unit-specific barcodes is used to analyze the number of units the pre-amplification targets were associated with. Such amplification and / or analysis steps, such as sequencing, may follow a hybrid capture step. For example, nucleic acids in a sample may be hybridized to targets associated with units in a hybrid capture step. One or more barcode sequences may be incorporated to hybridized nucleic acids, for example by a nucleic acid extension elongation reaction. The hybridized nucleic acids may be amplified and / or analyzed. The number of hybridized nucleic acids from the sample may be analyzed by counting the number of different barcodes. The identity of barcodes described herein may be probed using hybridization, sequencing, or any suitable method known in the art. Barcodes described herein may be generated through an oligomer synthesis method (such as nucleic acid synthesis or peptide synthesis), e.g. a number of rounds of monomer, (such as nucleotide) incorporation, such as random incorporation. In some embodiments, barcodes are generated through oligomer synthesis of, of about, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more rounds of monomer incorporation. Barcodes may be synthesized through oligomer synthesis wherein the number of rounds of monomer incorporation falls within any range bound by the foregoing values, e.g. 2-30 rounds, 8-10 rounds etc. In some embodiments, presynthesized barcodes attached to units. Barcodes described herein may comprise oligonucleotides. Synthesis of barcodes may be achieved through oligomer synthesis methods described herein. For example, units may be routed iteratively through reaction chambers or channels of the microfluidic devices described herein. One or more monomers (or dimers or longer building blocks) may be incorporated to the a nascent chain of a barcode in each routing cycle.

[0185] Cells may be bacterial cells or eukaryotic cells, for example cells derived from cell culture, animals, or human subjects, such as cells derived from patient samples. Droplets may be formed by the mixture of immiscible fluids, such as water and oil or other organic solvents, to form an emulsion. Droplet formation for use in microfluidic devices is described in U.S. Pat. Nos. 8,528,589, 9,364,803, 8,658,430, WO2014001781, and US20080286751, which are herein incorporated by reference in their entirety with respect to droplet formation in microfluidic devices.

[0186] The methods described herein can take advantage of beads or other types of units maintaining their order throughout an iterative modification process. In some embodiments, the beads or other types of units cannot pass each other or stick together. The bead or other type of unit distribution may be adjusted to be fairly monodisperse throughout the process. In some embodiments, units are passed through a size selection mechanism generating a population of units that substantially or entirely fall within a predesignated size range, for example by passing units through a size sorter. Units may be size sorted such that the likelihood of detected or undetected undesired unit mixing within the channels of the devices described herein, e.g. within channels having widths that physically prevent mixing of units of a selected average or nominal diameter and / or size, is minimized.

[0187] The beads or other types of units may swell when exposed to non-aqueous reagents, such as toluene, used in DNA synthesis. A swollen bead may stick to capillary walls and impede flow. Various materials, such as divinylbenzene (DVB) cross-linking of polymeric beads can mitigate swelling at an appropriate concentration. Introduction of surfactants may be used to reduce bead / unit adhesion. In more detail, one or more embodiments of the method(s) and / or system(s) described can implement beads or other types of units configured to be non-swelling, non-brittle, monodisperse, and / or porous, and devoid of outlier (in relation to morphological uniformity) beads or other types of units. Outlier units (e.g. beads may be removed by filtering, sieving, suspension processes, processes involving removal of a supernatant of smaller-than-desired particles, or by any suitable method known in the art. FIG. 36 depicts swelling response of divinylbenzene-cross-linked polystyrene (PS) beads in organic solvent versus percent divinylbenzene (DVB) cross-linking agent (in terms of molar ratio between PS and DVB). Beads were put in toluene to quantify swelling response of cross-linked beads in organic solvents. In various embodiments, swelling response of beads can be similarly measured in alternative solvents, such as acetonitrile, toluene, dichloromethane, tetrahydrofuran (THF), pyridine, N-methyl pyrrolidinone (NMP), 2,6-lutidine, carbon disulfide, 1,2-dichloroethane, 1,1-dichloroethane, chloroform, dimethylformamide, dimethylacetamide, dimethylsulfoxide (DMSO), ethylene carbonate, 1,4-dioxane, DME (1,2-dimethoxyethane), nitromethane, methyl tert-butyl ether, methyl ethyl ketone (butanone), or dichloromethane. Without being bound by theory, beads cross-linked in high cross-linker ratios may be brittle and / or be subject to surface solvation (e.g., as in the range from 60-80% DVB cross-linking in FIG. 36), while beads cross-linked in lower cross-linker ratios exhibit acceptable performance in terms of minimal swelling, brittleness, and solvation (e.g., as in the range from 30-60% DVB cross-linker in FIG. 36). Cross-linked beads may be functionalized by a variety of suitable moieties described elsewhere herein or otherwise known in the art, such as by amine or carboxyl groups.

[0188] In various embodiments, units may be filtered by size to provide a uniformly sized unit population. For example, units having a diameter greater than an upper threshold may be filtered out by a suitable method described herein or is known in the art. Amine functionalized polystyrene beads were cross-linked with 35% DVB. Cross-linked beads had a diameter of 34.4 μm with a coefficient of variation of 2.6%. Particles larger than 40 μm were removed by sieving through a 40 μm electroformed mesh. In some embodiments, particles smaller than a desired size are removed by a suitable method described herein or otherwise known in the art, such as by an electroformed mesh of appropriate size. Such size filtering methods may be applied on cross-linked beads of any suitable kind, which may be functionalized by one or more suitable moieties described elsewhere herein or otherwise known in the art, such as by amine or carboxyl groups. In a related variation, small particles, particle fragments, dust, and other contaminants may be removed by suspending the monodisperse PS beads in solution and letting the desired particles settle for a period of time, after which the supernatant with undesired particles and fragments may be removed. In another variation, small particles, particle fragments, dust, and other contaminants may be removed by centrifugation and removal of the supernatant with undesired particles and fragments. In any of the examples and variations described, one or more successive washing steps, optionally intercalated by other filtering steps, such as settling steps, may be implemented to remove undesired particles.

[0189] In embodiments, cross-linking can be performed using one or more of chemical cross-linking agents (e.g., DVB, glutaraldehyde, formaldehyde, epoxy compounds, dialdehyde, dichloroethane, etc.), radiation-induced cross-linking procedures, oxidative cross-linking procedures, and other cross-linking procedures.

[0190] In embodiments, beads used according to the methods described herein are crosslinked at a molar cross-linker ratio of, of about, or of greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher. Beads used according to the methods described herein may be crosslinked at a molar cross-linker ratio of less than 90%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or lower. Beads used according to the methods described herein may be crosslinked at a molar cross-linker ratio that falls within a range bounded by any of the foregoing values (e.g. 10-60%, 3-60%, 45-55%, etc.). Ranges of cross-linking and / or different types of cross-linking agents can be selected to tune swelling behavior, brittleness, and / or solvation as desired. For example, aforementioned parameters may be selected to limit swelling to, to about, or to less than 10%, 9% 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less.

[0191] Units, such as bead units can range in size according to the various embodiments described herein. For example, all or substantially all (e.g. more than 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.99%, 99.995%, 99.999% or more) units used in the methods and devices described herein may have a diameter and / or size from about, at least, or at least about 20 nm, 100 nm, 500 nm, 1000 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm. Those of skill in the art will appreciate that the unit diameter and / or size may have a value that falls within any range bound by any of these values, for example 20-100 nm, 100-500 nm, 500-1000 nm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm. The coefficient of variation for the size or cross-section of the units can be about, at least, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more. The coefficient of variation for the size or cross-section of the units can be about less than, or about less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. The units can also be oval. Droplet volume may be about, at least or at least about 10 femtoliters (fl), 100 fl, 1 pl, 10 pl, 100 pl, 500 pl, 1 nanoliter (nl), 10 nl, 50 nl, 100 nl, 300 nl, 400 nl, 500 nl, 600 nl, 700 nl, 800 nl, 900 nl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 50, pl, 100 μl, or more. The droplet volume may fall in a range bounded by any of the foregoing values, e.g. 10-100 femtoliters (fl), 100-1000 fl, 1-10 picoliters (pl), 10-100 pl, 100-500 pl, 500-1000 pl, 1-10 nanoliter (nl), 10-100 nl, 100-200 nl, 200-300 nl, 300-400 nl, 400-500 nl, 500-600 nl, 600-700 nl, 700-800 nl, 800-900 nl, 900-1000 nl, 1-10 μl, 10-50 μl, or 50-100 μl. Values for the unit or droplet size may range between any of the potential values set forth for the unit or droplet size herein.

[0192] In various embodiments, screening procedures may be applied to a library of products (or a subset thereof having selected properties) associated with units for which positional encoding is maintained. The positional encoding of the units can be used to identify products of interest. For example, after a library of products is made, the associated units, arranged in a 1d-array with known absolute or relative positions, can be exposed to a set of screening reagents. In various embodiments, screening reagents are delivered in the same or a similar manner as the reagents for modification procedures. Screening reagents may be moved through channels holding the products to be screened, such as channels holding the associated units in an ordered 1d-array. The units or the associated products may be evaluated for their reactivity to screening interactions, for example by optical analysis of the units in place or by flowing the units past a detector, such as an optical or magnetic detector. Units or associated products displaying features of interest, such as an ability to interact with a target compound, can be detected. A product associated with a unit detected for a screened feature can be identified, for example by the position of the unit.

[0193] In some embodiments, the physical encoding on the units may be associated with the units' positional encoding within a system. For example, the physical encoding of units may be read once in the beginning or end of one or more procedures within a system maintaining positional encoding and the physical and positional encodings of the units may be associated. This association between physical encodings and products can be used in downstream procedures even in the case where the positional encoding of the units is lost, for example when the units have been removed from an ordered 1d-array or otherwise disordered with respect to one another.Pumps

[0194] The systems and devices described in further detail elsewhere herein may contain pumps, for example for moving solutions or units through the channels of microfluidic devices, or for delivery of reagents into the reaction chambers of microfluidic devices. These pumps may be mechanical or non-mechanical, and utilize driving forces such as piezoelectrical, electrostatic, electro-osmotic, solenoid, thermo-pneumatic, pneumatic, magnetic, vacuum, or passive gravity or capillary forces, or other appropriate forces known to those of skill in the art (see Iverson B D et al, 2008, incorporated herein by reference in its entirety). The pumps may comprise peristaltic, syringe, vacuum, piezoelectric, or passive, or other appropriate pumps known to those of skill in the art. The pump may be connected to a flow rate sensor and a pressure controller.

[0195] In various embodiments, pumps are used for routing (e.g. steering units through several junctions, e.g. Y-junctions, to destination(s), e.g. a microfluidic destination, such as intone or more reaction chambers. The pump described herein may be used to generate pulses of about 100 pl over <10 msec. In some embodiments, pumps described herein are configured to generate pulses of about, at least, or at least about 1 pl, 2 pl, 3 pl, 4 pl, 5 pl, 6 pl, 7 pl, 8 pl, 9 pl, 10 pl, 20 pl, 30 pl, 40 pl, 50 pl, 60 pl, 70 pl, 80 pl, 90 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 1 nl, 2 nl, 3 nl, 4 nl, 5 nl, 6 nl, 7 nl, 8 nl, 9 nl, 10 nl, or more. The pumps described herein may be configured to generate less than or less than about 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 1000 nl, 900 nl, 800 nl, 700 nl, 600 nl, 500 nl, 400 nl, 300 nl, 200 nl, 100 nl, 90 nl, 80 nl, 70 nl, 60 nl, 50 nl, 40 nl, 30 nl, 20 nl, 10 nl, 9 nl, 8 nl, 7 nl, 6 nl, 5 nl, 4 nl, 3 nl, 2 nl, 1 nl, 900 pl, 800 pl, 700 pl, 600 pl, 500 pl, 400 pl, 300 pl, 200 pl, 100 pl, 90 pl, 80 pl, 70 pl, 60 pl, 50 pl, 40 pl, 30 pl, 20 pl, 10 pl, 9 pl, 8 pl, 7 pl, 6 pl, 5 pl, 4 pl, 3 pl, 2 pl, 1 pl or less. Those of skill in the art will appreciate that the pumps described herein may be used to generate pulses of a volume that falls within any range bound by any of these values, for example 10-50 pl, 10-100 pl, 50-100 pl, 100-200 pl, 200-300 pl, 300-400 pl, 400-500 pl, 500-600 pl, 600-700 pl, 700-800 pl, 800-900 pl, 900-1000 pl, or 1-10 pl. Values for the pulse volumes generated by the pumps described herein may range between any of the potential values set forth for the pulse volumes herein. In some embodiments, pumps described herein are configured to generate pulses of volumes described herein in over about, less than or less than about 500 msec, 400 msec, 300 msec, 200 msec, 100 msec, 90 msec, 80 msec, 70 msec, 60 msec, 50 msec, 40 msec, 30 msec, 25 msec, 20 msec, 10 msec, 9 msec, 8 msec, 7 msec, 6 msec, 5 msec, 4 msec, 3 msec, 2 msec, 1 msec, 0.9 msec, 0.8 msec, 0.7 msec, 0.6 msec, 0.5 msec, 0.4 msec, 0.3 msec, 0.2 msec, 0.1 msec or less.

[0196] Any appropriate pump can be used, including, but not limited to, electroosmotic pumps, which are cost-effective, compact, and can achieve high flow rates with a sub-millisecond response time.

[0197] A pump may be used to route (e.g. steer) units, e.g. microbeads via cross-flow routing. In some embodiments, the units within the microfluidics devices described herein are passed through a primary channel, which branches into at least two side channels, e.g. in a Y-shaped geometry. At least two additional cross-flow channels may be configured to intersect the main channel near the junction. As a unit, e.g. passes the cross-flow channels, a pulse of fluid may be delivered perpendicular to the unit's velocity. This pulse may be used to cause the unit to be deflected to the side of the channel. Laminar flow within the microfluidic devices described herein may be used to enable the unit to continue its forward motion in the same relative position in the channel adopted after deflection. Such deflected units may be caused to deterministically enter the side channel towards which it was deflected. FIG. 33 provides an illustration of a junction with cross-flow routing. Pneumatic pumps were used to move bead position laterally within laminar flow as individual beads were flowed from the left channel into the junction, thereby moving the beads' position within cross-section of the laminar flow toward one of the two branch channels on the right. Beads that were moved upward within the cross-section of the laminar flow followed a path into the upper branch channel and beads that were moved downward within the cross-section of the laminar flow followed a path into the lower branch channel.

[0198] FIG. 34B provides and exemplar electroosmotic pump. Electroosmotic pump elements of suitable configurations (such as a cylindrical frit 5 mm long and 1 mm in diameter) may be used to produce high pressures. In various embodiments, electroosmotic pump elements comprise a “pancake” design, for example one comprising a frit 5 mm in diameter and 1 mm long. Suitable pump elements may be selected in order to improve ability to prime, (such as by decreasing resistance to flow; to increase flow rates; and / or to improve ease of assembly. Any suitable method known in the art may be used to fabricate such electroosmotic pump elements.

[0199] Also provided herein is a high-speed pump, such as an electroosmotic high-speed pump, capable of sorting beads into reaction chambers. In various embodiments, such pumps may be configured to route units, e.g. beads, at, at about, or at least 1 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 42 Hz, 45 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, 5 kHz, 6 kHz, 7 kHz, 8 kHz, 9 kHz, 10 kHz, or greater. Such pumps may be configured to achieve such routing frequencies with an accuracy of, of about, or of at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.99%, 99.993%, 99.995% 99.999%, 99.9995% 99.9999% or greater. In some embodiments, the pump is configured to have a flow rate of, of about, or of at least 25 nl / s and / or a switching rate of, of about, or of at least 30 Hz for at least 1 hour. In some embodiments, the pump is configured to have a flow rate of, of about, or of at least 10 nL / s and / or rise times (t) of, of about, or of at least 10 msec, 9 msec, 8 msec, 7 msec, 6 msec, 5 msec, 4 msec, 3 msec, 2 msec, 1 msec, 900 μsec, 800 μsec, 700 μsec, 600 μsec, 500 μsec, 400 μsec, 300 μsec, 200 μsec, 100 μsec, 50 μsec, 10 μsec, 5 μsec, or 1 μsec, or less when integrated into a fluidic system.

[0200] In some embodiments, microfluidic devices comprising such pumps may be configured to result the synthesis of, of about, or of at least 10,000 oligonucleotides. Such oligonucleotides may be, be about, or be at least 5, 10, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10,000 nucleotides long, or longer. In various embodiments, microfluidic devices comprising such pumps may be used to synthesize such oligonucleotides in, in about, or in less than 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours or less. Such microfluidic devices may be configured to achieve such oligonucleotide synthesis with, with about, or with fewer than 1000, 500, 400, 300, 200, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30 20, 10, 5, 4, 3, 2 or fewer errors (i.e. units, such as beads, with an incorrect sequence).

[0201] In some embodiments, the pump has at least a single microfluidic bifurcation and manual or semi-manual switched routing, e.g. steering, of, of about, or of at least 100 particles with, with about or with at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.99%, 99.995%, or 100% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 100 individual particles at, at about, or at at least 1 Hz through a single bifurcation with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995%, or 100% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 1000 individual particles at, at about or at at least 5 Hz through two serial bifurcations into four outlets with, with about, or with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 1000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 individual particles at, at about or at least 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 200, 2500, 3000, 3500, 4000, 4500, or 5000 Hz through two serial bifurcations into at least one, two, or four outlets with, with about, or with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995% accuracy.

[0202] In some embodiments, the pump is a piezo or solenoid driven pump.

[0203] The mobile units may be in a fluid or solution. Pumps may be used to control the flow rate and / or pressure of the fluid and thereby control the flow rate of the units. Pumps may also be used to control the direction of the fluid or solution flow in the device and thereby control the flow direction of the unit. Changes in the flow direction of a fluid may be used to distribute the mobile units into secondary channels, branch channels, branch points, or reaction chambers. For example, a pump at the first end of a channel may apply a flow rate such that the units move down the channel to a branch point that branches into two, three, or more channels. The branch point may comprise a router, e.g. a distributor, or may not comprise a router. As the units approach the branch point, the pump at the first end of the channel is shut off or slowed, and a second pump at the end of one of the branch channels is turned on, resulting in flow of the fluid comprising the mobile units towards the second pump and down the chosen branch channel. Each branch channel may have a separate pump that can be controlled independently. Mobile units can be routed into the individual branch channels by turning on the appropriate pump for each branch channel as the unit approaches or passes through the branch point. Individual units or groups of units may be routed into branch channels.

[0204] Units in a fluid may be passed through the channels or the path of a detector at a flow rate of about, at least, or at least about 10 nl / min, 20 nl / min, 30 nl / min, 40 nl / min, 50 nl / min, 60 nl / min, 70 nl / min, 80 nl / min, 90 nl / min, 100 nl / min, 200 nl / min, 300 nl / min, 400 nl / min, 500 nl / min, 600 nl / min, 700 nl / min, 800 nl / min, 900 nl / min, 1 μl / min, 2 μl / min, 3 pl / min, 4 μl / min, 5 μl / min, 6 μl / min, 7 μl / min, 8 μl / min, 9 μl / min, 10 μl / min, 20 μl / min, 30 pl / min, 40 μl / min, 50 μl / min, 60 μl / min, 70 μl / min, 80 μl / min, 90 μl / min, 100 μl / min, or faster. In some cases, units in a fluid may be passed through the path of a detector at a flow rate of at most, or at most about 100 μl / min, 90 μl / min, 80 μl / min, 70 μl / min, 60 μl / min, 50 pl / min, 40 μl / min, 30 μl / min, 20 μl / min, 10 μl / min, 9 μl / min, 8 μl / min, 7 μl / min, 6 μl / min, 5 pl / min, 4 μl / min, 3 μl / min, 2 μl / min, 1 μl / min, 100 nl / min, 90 nl / min, 80 nl / min, 70 nl / min, 60 nl / min, 50 nl / min, 40 nl / min, 30 nl / min, 20 nl / min, 10 nl / min, or slower. Those of skill in the art appreciate that the flow rate may fall within any range bound by any of these values, for example 10-100 nl / min, 100-500 nl / min, or 500-1000 nl / min. Units and / or carrier fluid may also be passed through the device at a flow rate of about, at least, or at least about 0.1 cm / min, 0.5 cm / min, 1 cm / min, 2 cm / min, 3 cm / min, 4 cm / min, 5 cm / min, 6 cm / min, 7 cm / min, 8 cm / min, 9 cm / min, 10 cm / min, 20 cm / min, 30 cm / min, 40 cm / min, 50 cm / min, 60 cm / min, 70 cm / min, 80 cm / min, 90 cm / min, 1 m / min, 2 m / min, 3 m / min, 4 m / min, 5 m / min, 6 m / min, 7 m / min, 8 m / min, 9 m / min, 10 m / min, 20 m / min, 30 m / min, 40 m / min, 50 m / min, 60 m / min, 70 m / min, 80 m / min, 90 m / min, 100 m / min, or faster. In some cases, carrier fluid and / or units in a fluid may be passed through the channels or the path of a detector at a flow rate of at most, or at most about 100 m / min, 90 m / min, 80 m / min, 70 m / min, 60 m / min, 50 m / min, 40 m / min, 30 m / min, 20 m / min, 10 m / min, 9 m / min, 8 m / min, 7 m / min, 6 m / min, 5 m / min, 4 m / min, 3 m / min, 2 m / min, 1 m / min, 90 cm / min, 80 cm / min, 70 cm / min, 60 cm / min, 50 cm / min, 40 cm / min, 30 cm / min, 20 cm / min, 10 cm / min, 9 cm / min, 8 cm / min, 7 cm / min, 6 cm / min, 5 cm / min, 4 cm / min, 3 cm / min, 2 cm / min, 1 cm / min, 0.5 cm / min, 0.1 cm / min, or slower. Those of skill in the art appreciate that the carrier fluid and / or flow rate may fall within any range bound by any of these values, for example 10-100 cm / min, 100-500 cm / min, or 500-1000 cm / min. Values for the flow rate may range between any of the potential values set forth for the flow rate herein.

[0205] In various embodiments, pumps may be used to facilitate movement of mobile units. A pump may be attached to a channel to manipulate the flow rate of the fluid in the channel. The flow can be stopped, started, or the flow rate modulate via the speed of the pump, resulting in stopping, starting, or modulation of the unit movement through the device. Pump-controlled fluid flow may also be used to route, e.g. distribute, the mobile units by creating low pressure or vacuum conditions in the desired direction of travel for the mobile unit.

[0206] The methods and compositions described herein may be used to order units within a microfluidic device. Any suitable type of distributing algorithm can be used to distribute units in a first order into a second order. For example, units in a device may be distributed so that the correct units could be dispensed at the correct time or order. A first group of units may be dispensed followed by a second group of units and so on. In some embodiments, the exact order of the units within each such group is unimportant. Accordingly, units may be distributed so that the correct units are grouped into a first group of a desired size, a second group of a desired size etc. For example, the first group in a given grouping may have a size of 5 units whereas the second group in the grouping may have a size of 1 unit.Valves and Bead Stops

[0207] The device may contain elastomeric valves that close off sections of the channel(s). These valves may be mechanical or pressure-actuated. The valves may be deflected into or retracted from one channel or channel section in response to a force applied to another channel or channel section. The valves may be upwardly-deflecting, downwardly deflecting, side actuated, normally-closed, or some other type of valve. Elastomeric valves for use in microfluidic devices are described in US 20050072946, U.S. Pat. No. 6,408,878, US 20020127736, and U.S. Pat. No. 6,899,137, all which are herein incorporated by reference in their entirety, in particular with respect to the description of elastomeric valves. The device may have a combination of valve types. The valves may be operated by injecting gases, liquids, ionic solutions, or polymer solutions. A non-exclusive list of such solutions includes air, nitrogen, argon, water, silicon oils, perfluoropolyalkylether or other oils, salt solutions, polyethylene glycol, glycerol, and carbohydrates. Valves may also be operated by applying a vacuum to the channel(s).

[0208] The device may also contain valves that are physically separated from the reaction chamber(s) and / or branch channel(s). Reagents may be routed to the reaction chamber(s) and / or branch channel(s) via a delivery channel or an inlet directly or indirectly via a network of channels. In some embodiments, the delivery channel and / or inlet is about the same size or smaller than the reaction chamber(s), branch channel(s), and / or other channel(s) connecting the delivery channel and / or inlet to where reagents are designated to be delivered. In some embodiments, a delivery channel and / or an inlet interfaces with the reaction chamber(s), branch channel(s), and / or other connected channel(s) via a frit, a nozzle, a weir, a bead stop, or any other physical structure that enable fluid to pass through the structure but not units.

[0209] Valves and valve membranes can be constructed from any appropriate elastomeric material known in the art, including poly dimethylsiloxane (PDMS), polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicones. A non-exclusive list of elastomeric materials which may be utilized in connection with the present invention includes polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicone polymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F), perfluoropolyalkylether siloxane block copolymer, poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(1-butene), poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton), elastomeric compositions of polyvinylchloride (PVC), polysulfone, polycarbonate, polymethylmethacrylate (PMMA), and polytertrafluoro-ethylene (Teflon).

[0210] In some embodiments, the device includes one or more microfluidic check valves. A microfluidic check valve can be used to direct solution flow in only one direction through the valve. Any suitable check valve known in the art may be used in the systems and devices described herein.

[0211] Valve membranes separating flow channels may have a thickness of between about 0.01 and 1000 microns. Membrane thicknesses can be about, at least, or at least about 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm 7 μm, 8 μm, 9 μm 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm. Membrane thicknesses can be less than or less than about 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, 0.09 μm, 0.08 μm, 0.07 μm, 0.06 μm, 0.05 μm, 0.04 μm, 0.03 μm, 0.02 μm, 0.01 μm. Those of skill in the art will appreciate that the membrane thickness may have a size that falls within any range bound by any of these values, for example 0.01-0.1 μm, 0.1-1 μm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm. Values for the valve membrane thickness may range between any of the potential values set forth for the valve membrane thickness herein.

[0212] In some embodiments, the device described herein includes unit stops, such as a frit, wire, or weir. Unit stops may be used to halt the flow of single or multiple mobile units in one direction. Any appropriate unit stop known in the art may be used. Unit stops may be manufactured by inserting a wire within a channel, 3D printing a capillary connector that introduces a constriction or frit, and / or using photolithography to create a weir structure in a glass device or any suitable method known in the art. Unit stops may be used to halt the flow of single or multiple mobile units in one direction. The stopped mobile units may then be held, or the flow of the stopped mobile units may be reversed by altering the fluid flow or pressure e.g. via a pressure controller, pump, or vacuum. Unit stops may be used at any point in the device, such as at the beginning or end of a channel or branch channel, at a branch point, at the beginning or end of a reaction chamber, or any combination thereof.Detectors and Optical Detection Systems

[0213] The microfluidic devices described in various embodiments herein may include one or more detection systems for positionally tracking units within the microfluidic device. Each detection system may have one or more detectors. One or more detectors may be placed at any point in the device, for example to track units in a channel or the device, such as at any point in a channel or branch channel, before or after any or every branch point, before or after any or every router, e.g. distributor, before or after any or every reaction chamber, or before or after any or every outlet or inlet. One or more detectors may be used to ensure the correct number of units are distributed or steered into a channel or branch channel. In various embodiments, the one or more detectors are not be restricted to particular points or junctions of the systems described herein. For instance, the one or more detectors can be configured globally or regionally relative to a particular system. In one such embodiment, a charge-coupled device (CCD) / complementary metal oxide semiconductor (CMOS) or other wide-field imaging detector could monitor some or all of the units in a fluidic network of the system, or could be used to observe a region-of-interest (ROI) (e.g. to improve detection speed or reduce data volume). Additionally or alternatively, linear CCD arrays can be used to track beads in multiple channels (e.g., parallel channels, non-parallel channels). Additionally or alternatively, regional detectors, including but not limited to CCDs, can incorporate machine vision to identify and track beads as they move through the device. Additionally or alternatively, conductivity-based detection may be used to identify the presence and / or quantity of units, e.g. beads along a fluidic path between electrode probe points. In any embodiment, detectors may also be configured in any orientation relative to the channel(s) of the microfluidic devices described herein (e.g., above, below, laterally).

[0214] A detection system may be configured to execute steps for serial or parallel interrogation of the units using a variety of interrogatory devices, such as interrogatory devices using lasers or cameras, real time classification, and rapid, command driven distributing. The detection system may comprise a multiple part system, having, for example, one or more of a scanner that emits light at a particular excitation wavelength or set of wavelengths over the units in the microfluidic device, a detector that receives the emitted light or diffraction pattern from the units and converts it into a digital electrical signal that corresponds to the unit, a decoder that translates the signal into data which can then be sent to an associated computer for storage, and / or any other suitable component known in the art. Light illumination and detection devices may include fluorescence, surface plasmon resonance, total internal reflection fluorescence (TIRF), Raman spectroscopy, or any other suitable light illumination and / or detection technique known in the art. Detectors may include non-optical detectors such as magnetic detectors, conductivity sensors such as Coulter counters, capacitive sensors, dielectric spectroscopy, or any other non-optical detector known in the art, or any combination thereof. Multiple detectors, and multiple types or classes of detectors may be used in the device as described herein. For example, a device may have both one or more optical detectors and one or more non-optical detectors.

[0215] The detector may comprise a lamp (e.g. mercury, xenon, halogen), a laser (e.g. argon, krypton, helium neon, helium cadmium, diode laser), a light emitting diode (LED) or a diode laser coupled to a wavelength filter and a photon detector. The detector may also include a photomultiplier tube, a photodiode, or an avalanche photodiode. The detector may be optical fiber coupled or free-space optics coupled. The detector may also be a charge-coupled device (CCD) camera. Multiple detectors can be joined consecutively to read units that have multiple labels or to track a given unit through a device. Detectors configured to interrogate various locations within a device may collect information in parallel or in series.

[0216] Optical and non-optical detectors may detect and evaluate size, shape, orientation, positions, color, color spectra, interference patterns, barcode patterns, charge, magnetic or paramagnetic labels, or capacitance or conductivity of the units, or any combination thereof. Detectors may distinguish units from other non-unit elements such as dust, bubbles, unit fragments, or other contaminants. Detectors may be configured to collect location and speed information of units, which may be used for feedback control for the operation of the devices described herein, such as by increasing or decreasing the pressure of a carrier fluid to move the units, or to distribute the units. Detectors may be located in any channel, including without limitation a main channel, feeder channel, branch channel, reaction chamber or outlet channel and may be used to verify correct distributing or steering of the units, for example by determining the presence or absence of a unit, or by counting units to determine whether the correct number of units have been distributed or steered. Information collected by a detector may be used to identify an error in distributing and / or correct the distribution of a unit into the incorrect channel, as described in further detail elsewhere herein. As an example, a mis-distributed unit may be re-distributed into the correct channel, or a unit may be distributed into a channel to be held until it can be distributed into the correct channel.

[0217] An exemplary detector may comprise a single-mode or multimode source fiber and a receiver fiber placed adjacent or nearly adjacent to a channel. Such a detector is shown in FIG. 18. The source fiber provides an incident light and a receiver fiber receives light scattered or directed from the source fiber.

[0218] Highly accurate detection and counting of units can be achieved by using a detection system, such as an optical system to distinguish single units, even if closely spaced, from adjacent two (doubles), three (triplets), or more units (n-tuplets) as they traverse the detection system in the device. Two adjacent units (a double) can be distinguished from one or more units through a characteristic detection patterns, for example a detection pattern comprising a characteristic light transmission pattern as shown in FIG. 16A. Single, double, triple, and n-tuple units are shown to each result in a different characteristic signal shape that can be used to distinguish the number of units or beads passing through the detector. Detecting these characteristic patterns allows for positionally tracking the units or beads as they move through the device.

[0219] Complex combinations of single, double, triple, and n-tuple units can be distinguished by a detection system, including, without limitation, an optical detection system. Optical detection systems described herein may be used to analyze signal patterns of transmitted light, as shown in shown in FIG. 16A as units pass through a detection path as described previously or elsewhere herein. Single, double, triple, and n-tuple units traversing through an optical detection system may be identified by a characteristic intensity signal signature, including, without limitation, the characteristic “W” pattern obtained by single beads passing through the optical detection system described in Example 4. Characteristic signal patterns for two or more adjacent units may be established using the detection systems described herein. Signal patterns may be used to distinguish single, double, triple, and n-tuple units, including for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more adjacent units. Values for the adjacent units may range between any of the potential values set forth for the adjacent units herein. Detectors described herein may be used to detect and / or count units in a stacked configuration or unstacked configuration and can be used to count an arbitrarily large number of units. Based on the identification of a plurality of adjacent units, systems and methods described herein may be used to take an action on the plurality of adjacent beads. Such an action may include corrective mechanisms, including without limitation, directing one or more units, such as one or more of the detected adjacent units, to a holding chamber, applying a separating force on one or more units, such as one or more of the detected adjacent units, reprogramming of downstream directions of one or more units, such as one or more of the detected adjacent units, or combinations of one or more of the foregoing. One or more units, such as one or more beads that miss the application of designated application of reaction conditions, may be redirected immediately or at a later point such that the missed application of reaction conditions can be applied.

[0220] Without being bound by theory, the optical detection signals may be generated by the incident light being scattered (FIGS. 17A and 17B), and transmission intensity decreasing from baseline intensity as the leading edge of a first unit enters the optical path of an optical detection system (a) (FIG. 17B). Then, as the center of the first unit aligns with the optical path, the transmitted light momentarily increases, likely due to lensing of some, but not all of the light through the unit and into the receiving fiber (b). Transmitted light intensity decreases even further as the trailing edge of the first unit and leading edge of the second bead directly align with the optical path (c). Then, the transmitted light momentarily increases again, likely due to lensing as the center of the second unit aligns with the optical path (d). Transmitted light then momentarily decreases one last time as the trailing edge of the second unit traverses the optical path (e). This results in a characteristic “W” shape of the signal.

[0221] In various embodiments, methods and systems described herein are configured to distinguish bubbles from units in order to detect bubbles within the microfluidic devices described herein. Without being bound by theory, bubbles may interfere with device operation and / or cause miscounting of units. Bubbles traveling through a detector, such as an optical path lens, may cause a similar signal at the detector as that of a unit, for example a bead. In various embodiments, detectors, including without limitation, the optical detection systems described herein may be designed to distinguish bubbles from units using various characteristics. For example, bubbles may have a lower index of refraction than units, for example beads. The use of a sufficiently sensitive optical sensing system allows discrimination between the change in signal intensity from baseline caused by a bubble from that caused by a unit, for example a bead. In addition, a narrow size distribution of the units within the systems described herein reduces variation in unit signals, including for example the variation in signal width of a unit passing through the path of a detector at a selected speed. Without being bound by theory, greater bubble size variation can cause a greater variation in bubble signals. The combination of signal width variation and signal intensity differences can be combined to discriminate bubbles from other types of units in methods and systems described herein (FIGS. 18A and 18B).

[0222] Detectors may be configured to collect information from units passing the detector's path at a rate of about, at least, or at least about 1×10−1 units / sec (u / sec), 1×101 u / sec, 1×102 u / sec, 2×102 u / sec, 3×102 u / sec, 4×102 u / sec, 5×102 u / sec, 6×102 u / sec, 7×102 u / sec, 8×102 u / sec, 9×102 u / sec, 1×103 u / sec, 2×103 u / sec, 3×103 u / sec, 4×103 u / sec, 5×103 u / sec, 6×103 u / sec, 7×103 u / sec, 8×103 u / sec, 9×103 u / sec, 1×104 u / sec, 2×104 u / sec, 3×104 u / sec, 4×104 u / sec, 5×104 u / sec, 6×104 u / sec, 7×104 u / sec, 8×104 u / sec, 9×104 u / sec, 1×105 u / sec, 2×105 u / sec, 3×105 u / sec, 4×105 u / sec, 5×105 u / sec, 6×105 u / sec, 7×105 u / sec, 8×105 u / sec, 9×105 u / sec, 1×106 u / sec, 2×106 u / sec, 3×106 u / sec, 4×106 u / sec, 5×106 u / sec, 6×106 u / sec, 7×106 u / sec, 8×106 u / sec, 9×106 u / sec, 1×107 u / sec, 2×107 u / sec, 3×107 u / sec, 4×107 u / sec, 5×107 u / sec, or more. In some cases, detectors may be configured to collect information from units passing through the detector's path at a rate of at most, or at most about 5×107 u / sec, 4×107 u / sec, 3×107 u / sec, 2×107 u / sec, 1×107 u / sec, 9×106 u / sec, 8×106 u / sec, 7×106 u / sec, 6×106 u / sec, 5×106 u / sec, 4×106 u / sec, 3×106 u / sec, 2×106 u / sec, 1×106 u / sec, 9×105 u / sec, 8×105 u / sec, 7×105 u / sec, 6×105 u / sec, 5×105 u / sec, 4×105 u / sec, 3×105 u / sec, 2×105 u / sec, 1×105 u / sec, 9×104 u / sec, 8×104 u / sec, 7×104 u / sec, 6×104 u / sec, 5×104 u / sec, 4×104 u / sec, 3×104 u / sec, 2×104 u / sec, 1×104 u / sec, 9×103 u / sec, 8×103 u / sec, 7×103 u / sec, 6×103 u / sec, 5×103 u / sec, 4×103 u / sec, 3×103 u / sec, 2×103 u / sec, 1×103 u / sec, 9×102 u / sec, 8×102 u / sec, 7×102 u / sec, 6×102 u / sec, 5×102 u / sec, 4×102 u / sec, 3×102 u / sec, 2×102 u / sec, 1×102 u / sec, 1×101 u / sec, 1×10−1 u / sec or less. Those of skill in the art appreciate that the unit passing rate may fall within any range bound by any of these values, for example 1×102-1×103 u / sec, 1×103-5×103 u / sec, or 5×103-1×104 u / sec. Values for the information collection rate may range between any of the potential values set forth for the information collection rate herein.Nucleic Acid Synthesis

[0223] In one embodiment, the synthesis of large library of specific DNA or other nucleic acid molecules is achieved according to the methods and compositions described herein. A set of units begin in a primary channel and are directed according to a preassigned program to one of four distinct channels. Direction into these channels may be achieved by a multiway distributor, by two sequential bifurcations and corresponding two-way distributors, or by any other suitable method known in the art. Reagents, such as various phosphoramidites may be delivered to the channels. The units may be combined maintaining their positional encoding and reassigned and delivered into one of the four distinct channels. Accordingly, nucleotides may be added in iterative steps to a nascent chain on each unit.

[0224] In various embodiments, nucleic acid synthesis is performed in or on the units described herein within the microfluidic devices described herein. In some cases, nucleic acid synthesis is achieved using the phosphoramidite method. Alternative nucleic acid synthesis methods may also be used, such as H-phosphonate, phosphate triester, phosphodiester, phosphotriester, and phosphite triester methods. A non-exclusive list of reagents for these methods that may be delivered to the units comprises nucleotide phosphoramidite monomers; non-nucleoside phosphoramidite monomers; B-cyanoethyl; 4,4′-dimethoxytrityl (DMT); tricholroacetic acid and / or dochloroacetic acid; an acedic azole catalyst, such as 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or other similar compounds; acetic anhydride, 1-methylimidazole, and / or DMAP; iodine; water; a weak base such as pyridine, lutidine, or collidine; tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO); 3-(Dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, and / or N,N,N′N′-Tetraethylthiuram disulfide; and controlled porous glass. Reagents for nucleic acid synthesis are available from purchase from numerous commercial sources, including American International Chemical (Natick Mass.), BD Biosciences (Palo Alto Calif.), and others. The specific reagents used may vary depending on the method of nucleic acid synthesis, e.g phosphoramidite or non-phosphoramidite reactions.

[0225] In some embodiments, nucleotides with suitable modifications for phosphoramidite or non-phosphoramidite chemistry are deposited on a functionalized unit(s) in the device. These nucleotides can be mononucleotides, dinucleotides, or longer oligonucleotides. Phosphoramidite-based nucleic acid synthesis chemistry typically involves the following steps in order: 1) coupling, 2) capping, 3) oxidation and / or sulfurization, 4) deblocking, and 5) desalting. Either oxidation or sulfurization may be used as one of the steps. Successive rounds of chemistry performed in the device may result in step-wise synthesis of high-quality polymers on units. In various embodiments, units described herein are subjected to one or more steps of nucleic acid synthesis in the microfluidic devices described herein. For example, one or more units in a reaction chamber may be contacted with reagents and solutions through one or more reagent channels that connect to the reaction chamber.

[0226] Methods of quickly synthesizing n-mer, such as about or at least about 100-, 150-, 200, 250-, 300, 350-, or longer nucleotide, oligonucleotides on units is further described herein in various embodiments. Such methods can use units functionalized with a chemical moiety suitable for nucleotide coupling. In various embodiments, the surface of the units described herein is chemically modified to provide a proper site for the linkage of a growing oligomer to the surface.

[0227] In some embodiments, a trialkoxysilyl amine (e.g. (CH3CH20)3Si—(CH2)2-NH2) is allowed to react with glass or silica surface SiOH groups, followed by reaction with succinic anhydride with the amine to create an amide linkage and a free OH on which the nucleotide chain can grow. In some embodiments, beads, such as polymeric beads e.g. polystyrene beads or divinylbenzene-cross-linked polystyrene beads comprise amino functionalization, hydroxyl functionalization, or other suitable functionalization known in the art.

[0228] In some embodiments, photocleavable linkers are used. A photocleavable linker may allow for a synthesized oligonucleotide to be removed from the units (e.g. by irradiation with light, e.g. ˜350 nm light) without cleaving the protecting groups on the nitrogenous functionalities on each base. The use of photocleavable linkers of this sort is described at www.glenresearch.com. Various other suitable cleavable linker groups are known in the art and may alternatively be used.

[0229] In some embodiments, adenine, guanine, thymine, cytosine, or uridine building blocks, or analogs / modified versions thereof are used as described in further detail elsewhere herein. In some cases, the added building blocks comprise dinucleotides, trinucleotides, or longer nucleotide based building blocks, such as building blocks containing about or at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. In some embodiments, large libraries of n-mer oligonucleotides are synthesized in parallel on units, e.g. about or at least 100, 1000, 10000, 100000, 1000000, 2000000, 3000000, 4000000, 5000000 units hosting oligonucleotide synthesis. Individual units may host synthesis of oligonucleotides that are different from each other.

[0230] A common method for the preparation of synthetic nucleic acids is based on the fundamental work of Caruthers and is known as the phosphoramidite method (M. H. Caruthers, Methods in Enzymology 154, 287-313, 1987; incorporated herein by reference in its entirety).

[0231] In some embodiments, the synthesis of DNA oligomers by the methods of the invention is achieved through phosphoramidite chemistry, reviewed in Streyer, Biochemistry (1988) pp 123-124 and U.S. Pat. No. 4,415,732, herein incorporated by reference. In various embodiments, the chemical synthesis of nucleic acids is performed using variations of the phosphoramidite chemistry developed for solid surfaces (Beaucage S L, Caruthers M H. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 1981; 22: 1859-1862; Caruthers M H. Gene synthesis machines—DNA chemistry and its uses. Science. 1985; 230:281-285., both of which are incorporated herein by reference in their entirety). For instance, phosphoramidite based methods can be used to synthesize abundant base, backbone and sugar modifications of deoxyribo- and ribonucleic acids, as well as nucleic acid analogs (Beaucage S L, Iyer R P. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron. 1992; 48:2223-2311; Beigelman L, Matulic-Adamic J, Karpeisky A, Haeberli P, Sweedler D. Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies. Methods Enzymol. 2000; 317:39-65; Chen X, Dudgeon N, Shen L, Wang J H. Chemical modification of gene silencing oligonucleotides for drug discovery and development. Drug Discov. Today. 2005; 10:587-593; Pankiewicz K W. Fluorinated nucleosides. Carbohydrate Res. 2000; 327:87-105; Lesnikowski Z J, Shi J, Schinazi R F. Nucleic acids and nucleosides containing carboranes. J. Organometallic Chem. 1999; 581: 156-169; Foldesi A, Trifonova A, Kundu M K, Chattopadhyaya J. The synthesis of deuterionucleosides. Nucleosides Nucleotides Nucleic Acids. 2000; 19: 1615-1656; Leumann C J. DNA Analogues: from supramolecular principles to biological properties. Bioorg. Med. Chem. 2002; 10:841-854; Petersen M, Wengel J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 2003; 21:74-81; De Mesmaeker A, Altmann K-H, Waldner A, Wendebom S. Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr. Opin. Struct. Biol. 1995; 5:343-355), all of which are incorporated herein by reference in their entirety.

[0232] In various embodiments, nucleic acids, e.g. nucleic acid double strands, are synthesized and / or assembled while attached on a unit. Methods of preparation of nucleic acid on a common solid support, are discussed in U.S. Pat. No. 7,790,369 and Pub. No. 2007-0087349, both of which are herein incorporated by reference in their entirety.Oligonucleotides

[0233] As used herein, the terms “preselected sequence”, “predefined sequence” or “predetermined sequence” are used interchangeably. The terms mean that the sequence of the polymer is known and chosen before synthesis or assembly of the polymer. Various aspects of the invention are described herein primarily with regard to the preparation of nucleic acids molecules, the sequence of the oligonucleotide or polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules. In one embodiment, oligonucleotides are short nucleic acid molecules. For example, oligonucleotides may be from about 10 to about 300 nucleotides, from about 20 to about 400 nucleotides, from about 30 to about 500 nucleotides, from about 40 to about 600 nucleotides, or more than about 600 nucleotides long. Those of skill in the art appreciate that the oligonucleotide lengths may fall within any range bounded by any of these values (e.g., from about 10 to about 400 nucleotides or from about 300 to about 400 nucleotides etc.). Suitably short or long oligonucleotides may be used as necessitated by the specific application. Individual oligonucleotides may be designed to have a different length from another in a library. Oligonucleotides can be relatively short, e.g. shorter than 200, 100, 80, 60, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, or 4 nucleotides, more particularly. Relatively longer oligonucleotides are also contemplated; in some embodiments, oligonucleotides are longer than or equal to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 500, 600 nucleotides, or longer. In some embodiments, oligonucleotides are single-stranded DNA or RNA molecules.

[0234] In some embodiments, oligonucleotides described herein are associated to the units described herein through an attachment via the oligonucleotides' 5′ end. In some embodiments, oligonucleotides described herein are associated to the units described herein through an attachment via the oligonucleotides' 3′ end. In some embodiments, the attachment is through a covalent bond or an affinity binding pair. In some embodiments, a second strand complementary to a first strand associated with a unit is synthesized. In some embodiments, the second strand is attached to the unit via its 3′ end. In some embodiments, the second strand is attached to the unit via its 5′ end. In some embodiments, oligonucleotides associated with a unit have a free 3′ end. In some embodiments, oligonucleotides associated with a unit have a free 5′ end. Oligonucleotides described under this paragraph may be synthesized using any synthesis method described herein or any suitable method known in the art.

[0235] In some embodiments, oligonucleotides attached to a unit are inverted to reverse the orientation with respect to the units, e.g. from 3′-bound to 5′-bound. Oligonucleotides described herein may be attached to units described herein through a cleavable linker, e.g. a cleavable linker arm of a branched linker attached to a unit. In various embodiments, a branched linker is attached to units described herein. Such branched linkers may comprise a first branch, e.g. a first branch comprising a first alkyne and a second branch, e.g. a second branch comprising a cleavable linker. An oligonucleotide may be attached to the cleavable linker via a first end, e.g. via its 3′ end. The other end, e.g. the 5′ end, of the oligonucleotide, may be attached to an azide group. In some embodiments, the oligonucleotide is circularized through the branched linker, e.g. by binding the free end of the oligonucleotide to the second branch. In some embodiments, the circularization is achieved by Cu(I) Click chemistry. For example, the azide group on the free end of the oligonucleotide may be reacted with the alkyne on the first branch of the branched linker. Upon circularization, the cleavable linker may be cleaved, e.g. using standard deprotection conditions, such as treatment with NH4OH at 55° C. for 15 hours, thereby releasing the first end, e.g. 3′ end of the oligonucleotide. Methods relating to inversion of oligonucleotides on solid supports are further discussed in U.S. Pat. Pub. 2017 / 0050162, which is incorporated herein by reference in its entirety.

[0236] In one aspect of the invention, a device for synthesizing a plurality of nucleic acids having a predetermined sequence is provided. The device can include units as described in further detail herein having a plurality of oligonucleotides. In some embodiments, the oligonucleotides are linked through their 3′ end to the units described herein. Yet, in other embodiments the oligonucleotides are linked through their 5′ end to the units described herein. Oligonucleotide linkages may be in a variety of forms, such covalent linkages or linkages comprising affinity binding.

[0237] An oligonucleotide may be immobilized on the units described herein via a nucleotide sequence (e.g., a degenerate binding sequence), a linker or spacer (e.g., a moiety that is not involved in hybridization). In some embodiments, the oligonucleotide comprises a spacer or linker to separate the oligonucleotide sequence from the unit. Useful spacers or linkers include photocleavable linkers, or other traditional chemical linkers. In one embodiment, oligonucleotides may be attached to a unit through a cleavable linkage moiety. For example, the unit may be functionalized to provide cleavable linkers for covalent attachment to the oligonucleotides. The linker moiety may be of six or more atoms in length. Alternatively, the cleavable moiety may be within an oligonucleotide and may be introduced during synthesis. A broad variety of cleavable moieties are available in the art of oligonucleotide synthesis (see e.g., Pon, R., Methods Mol. Biol. 20:465-496 (1993); Verma et al, Annu. Rev. Biochem. 67:99-134 (1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728). A suitable cleavable moiety may be selected to be compatible with the nature of the protecting group of the nucleoside bases, the choice of unit material, and / or the mode of reagent delivery, among others. In an exemplary embodiment, the oligonucleotides cleaved from the unit contain a free 3′-OH end. Alternatively, the free 3′-OH end may also be obtained by chemical or enzymatic treatment, following the cleavage of oligonucleotides. In various embodiments, the invention relates to methods and compositions for release of unit bound oligonucleotides into solution. The cleavable moiety may be removed under conditions which do not degrade the oligonucleotides. The linker may be cleaved using a variety of approaches, for example simultaneously under the same conditions as a deprotection step or subsequently utilizing a different condition or reagent for linker cleavage after the completion of a deprotection step.

[0238] As used herein, the term “duplex” may refer to a nucleic acid molecule that is at least partially double-stranded. The terms “nucleoside” or “nucleotide” may refer to those moieties which contain not only the conventional purine and pyrimidine bases, i.e., adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U), but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles or any other suitable modifications described herein or otherwise known in the art. In addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0239] As used herein, the terms “nucleoside” and “nucleotide” may refer to protected forms of nucleosides and nucleotides, e.g., wherein the base is protected with a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl, and purine and pyrimidine analogs.

[0240] Suitable nucleotide and nucleoside analogs will be known to those skilled in the art. Common analogs include, but are not limited to, 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methyl guanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylanminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxy acetic acid, uracil-5-oxy acetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine.

[0241] As used herein, the term “oligonucleotide” shall include poly deoxynucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones (for example PNAs). Thus, these terms include known types of oligonucleotide modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). There is no intended distinction in length between the term “polynucleotide” and “oligonucleotide,” and these terms may be used interchangeably.

[0242] The term “attached,” as in, for example, a unit having a moiety “attached” thereto, includes covalent binding, adsorption, and physical immobilization. The terms “binding” and “bound” are identical in meaning to the term “attached.

[0243] Oligonucleotides on units may be cleaved from their solid surface and optionally pooled to enable new applications such as, gene assembly, nucleic acid amplification, sequencing libraries, shRNA libraries etc. (Cleary M A, Kilian K, Wang Y Q, Bradshaw J, Cavet G, Ge W, Kulkami A, Paddison P J, Chang K, Sheth N, et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nature Methods. 2004; 1:241-248), gene synthesis (Richmond K E, Li M H, Rodesch M J, Patel M, Lowe A M, Kim C, Chu L L, Venkataramaian N, Flickinger S F, Kaysen J, et al. Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res. 2004; 32:5011-5018; Tian J D, Gong H, Sheng N J, Zhou X C, Gulari E, Gao X L, Church G. Accurate multiplex gene synthesis from programmable DNA microchips. Nature. 2004; 432: 1050-1054) and site-directed mutagenesis (Saboulard D, Dugas V, Jaber M, Broutin J, Souteyrand E, Sylvestre J, Delcourt M. High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips. BioTechniques. 2005; 39:363-368), all of which are herein incorporated by reference in their entirety. In some embodiments, reactions comprising such oligonucleotides may be performed without detaching the oligonucleotide from its unit.”Other Oligomers

[0244] In various embodiments, the invention relates to the synthesis, such as chemical synthesis, of molecules other than nucleic acids. The terms “peptide,”“peptidyl” and “peptidic” as used throughout the specification and claims are intended to include any structure comprised of two or more amino acids. The peptides synthesized according to the methods described herein may comprise about 5 to 10,000 amino acids, preferably about 5 to 1000 amino acids. The peptides synthesized according to the methods described herein may comprise, comprise about or comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 amino acids or more. The peptides synthesized according to the methods described herein may comprise a number of amino acids falling within the range bounded by any of the foregoing values, such as 2-900, 90-10,000, etc. The amino acids forming all or a part of a peptide may comprise any of the twenty conventional, naturally occurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y) or amino acids, e.g. non-naturally occurring amino acids. The term “non-conventional amino acid” refers to amino acids other than conventional amino acids, and includes, for example, isomers and modifications of the conventional amino acids (e.g., D-amino acids), non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine), and peptides having the naturally occurring amide —CONH— linkage replaced at one or more sites within the peptide backbone with a non-conventional linkage such as N-substituted amide, ester, thioamide, retropeptide (—NHCO—), retrothioamide (—NHCS—), sulfonamido (—S02NH—), and / or peptoid (N-substituted glycine) linkages. Accordingly, the peptidic molecules of the array include pseudopeptides and peptidomimetics. The peptides of this invention can be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.

[0245] The term “oligomer” may encompass any polynucleotide or polypeptide or other chemical compound with repeating moieties such as nucleotides, amino acids, carbohydrates and the like.

[0246] In some examples, all or some of the units in a group of units, e.g. units within a microfluidic device described herein each are attached to a different composition, such as a different oligonucleotide.Amplification of Nucleic Acids

[0247] In various embodiments, the methods and systems relate to amplification of single stranded nucleic acids.

[0248] The single stranded nucleic acids may be amplified using adaptors incorporated into the target sequence. Polymerase chain reaction in conjunction with primers corresponding to these adaptors, or any amplification method described herein or any other suitable amplification method known in the art, can be used to amplify the target.

[0249] The single stranded nucleic acids may be circularized upon hybridization with an adaptor. A single stranded nucleic acid may be circularized by joining its 5′ and 3′ ends, forming a contiguous circle. Various ligation methods and enzymes are suitable for the reaction as described elsewhere herein and otherwise known in the art. Circularized nucleic acids may be attached to units described herein. In some embodiments, nucleic acids are circularized while being associated with, e.g. attached to, units. For example, a unit may be isolated within a microfluidic device, for example, in a channel or chamber of a microfluidic device, nucleic acids associated with the unit may be released from the unit may be circularized, e.g. through the use of an adaptor. In some embodiments, the circularized nucleic acids are attached to the unit, e.g. covalently or through affinity binding.

[0250] Adaptors, according to the various embodiments of the invention, can be extended using the circularized single stranded nucleic acid as a template. Alternatively, one or more different primers may be used to anneal elsewhere on the circle in addition or instead of the adaptor and can be extended with a polymerase enzyme. The extension reaction, such as rolling circle amplification, multi-primer rolling circle amplification or any other suitable extension reaction, can facilitate the creation of one long and linear single stranded amplicon nucleic acids comprising alternating replicas of the single stranded template nucleic acid and the adaptor hybridization sequences. In some embodiments, the combined replicas of the adaptor hybridization sequences are copies of the adaptor sequence, or differ by less than 8, 7, 6, 5, 4, 3, or 2 nucleotides. These sequences will together be referred to as “adaptor copies” for ease, but it is understood that they may refer to a number of different types of sequences generated from the extension reaction using the circle as a template. Nucleotide extension products produced using templates described herein, such as circularized single stranded nucleic acid templates, may be attached to units described herein. In some embodiments, nucleic acid extension products are attached to the same unit as the template nucleic acid that is used as a template to produce the nucleic acid extension product.

[0251] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved using the methods described herein or by any suitable method known in the art. For example, extension and / or amplification products may be cleaved at the 5′ end of a recognition site by Type II endonucleases. The cutting site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more upstream from the first nucleotide of the recognition site. The 5′ or 3′ end of a recognition site may form a 0-, 1-, 2-, 3-, 4-, or 5-nucleotide overhang. Blunt Type II endonucleases which cleave with a 0-nucleotide overhang include Mlyl and Schl. Exemplary Type IIS endonucleases which generate 5′ overhangs (e.g., 1, 2, 3, 4, 5 nucleotides overhangs) include, but are not limited to, Alwl, Bed, BceAI, BsmAI, BsmFI, Fokl, Hgal, Plel, SfaNI, BfuAI, Bsal, BspMI, BtgZI, Earl, BspQI, Sapl, Sgel, BceFI, BslFI, BsoMAI, Bst71I, Faql, Acelll, BbvII, Bvel, and Lgul. Nicking endonucleases which remove the recognition site and cleave on the 5′ site of the recognition site include, but are not limited to Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, Finl, Tsui, UbaFl 1I, Unbl, Vpakl 1AI, BspGI, Drdll, Pfll 108I, and UbaPI.

[0252] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved by non-Type IIS endonucleases which cleave at the 5′ end of the recognition site on both strands to generate a blunt end. The amplification product may be cleaved by non-Type IIS endonucleases which cleave at the 5′ end of the recognition site on one strand and in the middle of the recognition site on the other strand, generating a 5′ overhang. Examples of endonucleases which generate a 5′ overhang include, but are not limited to, BfuCI, DpnII, Fatl, Mbol, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I, Tsp45I, Aoxl, BscFI, Bspl43I, BssMI, BseENII, BstMBI, Kzo9I, Nedll, Sse9I, Tasl, TspEI, Ajnl, BstSCI, EcoRII, Maelll, NmuCI, and Psp6I.

[0253] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved by nicking endonucleases which cleave at the 5′ end of a recognition site to produce a nick. The nicking site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more upstream from the first nucleotide of the recognition site. Exemplary nicking endonucleases include, but are not limited to, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, Finl, Tsui, UbaF 1I, Unbl, Vpakl 1AI, BspGI, Drdll, Pfll 108I, and UbaPI.

[0254] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved at the 3′ end of a recognition site by Type IIS endonucleases. The 5′ or 3′ end of a recognition site may form a 0-, 1-, 2-, 3-, 4-, or 5-nucleotide overhang. The cutting site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more downstream from the last nucleotide of the recognition site. Type IIS endonucleases which cleave at 0 nucleotides downstream of the last nucleotide of the recognition site include Mlyl and Schl. Exemplary Type IIS endonucleases which generate 3′ overhangs (e.g., 1, 2, 3, 4, 5 nucleotide overhangs) include, but are not limited to, Mnll, BspCNI, Bsrl, BtsCI, Hphl, HpyAV, MboII, Acul, BciVI, Bmrl, Bpml, BpuEI, BseRI, Bsgl, Bsml, BsrDI, Btsl, Ecil, Mmel, NmeAIII, Hin4II, TscAI, Bce83I, Bmul, Bsbl, and BscCI. Non-Type II endonucleases which remove the recognition site on one strand and generate a 3′ overhang or blunt end on the other strand include, but are not limited to Nlalll, Hpy99I, TspRI, Fael, Hinlll, Hsp92II, Setl, Tail, Tscl, TscAI, and TseFI. Nicking endonucleases which remove the recognition site and cut on the 3′ end of the recognition site include Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI.

[0255] The adaptor sequences described herein may comprise one or more restriction recognition sites. In some embodiments, the recognition site is at least 4, 5, or 6 base pairs long. In some embodiments, the recognition site is non-palindromic. In some embodiments, the adaptor oligonucleotide comprises two or more recognition sites. Two or more recognition sites may be cleaved with one or more restriction enzymes. Exemplary pairs of recognition sites in an adaptor sequence include, but are not limited to, Mlyl-Mlyl, Mlyl-Nt.AlwI, Bsal-Mlyl, Mlyl-BciVI, and BfuCI-Mlyl.Sequencing

[0256] In any of the embodiments, the detection or quantification analysis of polynucleotides described herein, e.g. polynucleotides synthesized on units described herein or polynucleotides captured thereby or produced based thereon, such as amplification products using such polynucleotides as templates, can be accomplished by sequencing. The subunits or entire synthesized oligonucleotides can be detected via full sequencing of all oligonucleotides by any suitable methods known in the art, e.g., Illumina HiSeq 2500, including the sequencing methods described herein.

[0257] Sequencing can be accomplished through classic Sanger sequencing methods which are well known in the art. Sequencing can also be accomplished using high-throughput systems some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, i.e., detection of sequence in real time or substantially real time.

[0258] In some embodiments, high-throughput sequencing involves the use of next-generation sequencing (NGS) technology available by Illumina Genome Analyzer II, MiSeq personal sequencer, or HiSeq systems, such as those using HiSeq 4000, HiSeq 3000, HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000.

[0259] In some embodiments, NGS comprises the use of technology available by ABI SOLiD System. This genetic analysis platform may be used for massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology may utilize sequential ligation with dye-labeled oligonucleotides.

[0260] The NGS may comprise ion semiconductor sequencing (e.g., using technology from formerly Life Technologies, now Thermo Fisher (Ion Torrent)). Ion semiconductor sequencing can take advantage of the fact that when a nucleotide is incorporated into a strand of DNA, an ion can be released.

[0261] In some embodiments, NGS comprises the use of the Single Molecule Sequencing by Synthesis (SMSS) method, discussed in further detail in U.S. Pub. Nos. 2006 / 002471 I; 2006 / 0024678; 2006 / 0012793; 2006 / 0012784; and 2005 / 0100932.

[0262] High-throughput sequencing of oligonucleotides can be achieved using any suitable sequencing method known in the art, such as those commercialized by Pacific Biosciences, Complete Genomics, Genia Technologies, Halcyon Molecular, Oxford Nanopore Technologies and the like. The sequencing of polynucleotides may be performed using a next generation sequencing technique, e.g. real-time (SMRT™) technology by Pacific Biosciences. In some embodiments, the next generation sequencing comprises nanopore sequencing (See e.g., Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001). The nanopore sequencing technology from Oxford Nanopore Technologies; e.g., a GridlON system is used in various embodiments. Nanopore sequencing technology from Roche (formerly GENIA) can be used. The next generation sequencing may comprise DNA nanoball sequencing (as performed, e.g., by Complete Genomics; see e.g., Drmanac et al. (2010) Science 327: 78-81).Genes

[0263] The methods and compositions of the invention in various embodiments allow for the construction of gene libraries comprising a collection of individually accessible polynucleotides of interest. The polynucleotides can be linear, can be maintained in vectors (e. g., plasmid or phage), cells (e. g., bacterial cells), as purified DNA, or in other suitable forms known in the art. Library members (variously referred to as clones, constructs, polynucleotides, etc.) can be stored in a variety of ways for retrieval and use, including for example, in multiwell culture or microtiter plates, in vials, in a suitable cellular environment (e.g., E. coli cells), as purified DNA compositions on suitable storage media (e.g., the Storage IsoCodeD ID™ DNA library card; Schleicher & Schuell Bioscience), or a variety of other suitable library forms known in the art. A gene library may comprise about or at least 10, 100, 200, 300, 400, 500, 600, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7500, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 75000, 100000 members, or more.

[0264] In various embodiments, the methods and compositions of the invention allow for production of synthetic (i.e. de novo synthesized) genes. Libraries comprising synthetic genes may be constructed by a variety of methods described in further detail elsewhere herein, such as PCA, non-PCA gene assembly methods or hierarchical gene assembly, combining (“stitching”) two or more double-stranded polynucleotides (referred to here as “synthons”) to produce larger DNA units (i.e., multisynthons or chassis). Libraries of large constructs may comprise polynucleotides that are about or at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb long or longer. The large constructs may be shorter than 50000, 20000, 10000 or 5000 base pairs. The synthesis of any number of polypeptide-segment encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, as well as non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest is contemplated according to the embodiments of the invention. The term “gene” as used herein may refer broadly to any type of coding or non-coding, long polynucleotide or polynucleotide analog.

[0265] In some embodiments, nucleic acids and / or nucleic acid libraries described herein comprise genes encoding for a part or all of the genome of a synthetic organism, e.g. a virus or a bacterium. The terms “gene”, “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a nucleotide polymer. Unless otherwise limited, the same include known analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides. They can be of polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more of the modified nucleotides described herein or any other modified nucleotides known in the art, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0266] The term nucleic acid encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0267] The term nucleic acid also encompasses any chemical modification thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrosta...

Examples

example 1

Positional Encoding Device Architecture

[0445]We constructed a system configured to perform loading, holding, and manipulating of units as an example of positional encoding within a microfluidic device. The system comprises a fluidic network and a flow control system that controls the fluid flow through the network, as depicted in FIG. 14. The fluidic network is constructed from fused silica capillaries (363 um OD, 50 um ID, Molex), capillary tubing connectors (CapTight connectors, LabSmith), and custom fabricated connectors.

[0446]The bead-containing portion of the network begins with a feeder channel 1405 that serves as both a loading channel and a repository for beads prior to bead rearrangement. This channel was connected to a main channel 1410 through a custom-fabricated T-connector 1406 that serves as a bead spacer. Two branch channels 1412, 1420 were connected to the main channel via additional T-connectors that were configured to service as bead spacers. Beads may be distribut...

example 2

Positional Encoding Device—Bead Spacer

[0450]We first manually loaded a set of highly monodisperse 40 μm beads into the feeder channel 1405, capped the channel input with a bead stop 1404, and connected the other side of the bead stop to the channel's fluid control line 1403. Then, we directed flow in the main channel toward the top side of the main channel 1410, 1418, 1426 and applied pressure to the feeder channel via the reservoir 1416 and the main channel reservoir 1417.

[0451]Beads were fed through the feeder channel in a stacked regime. When abutting beads reached the T-connector, the cross-flow created separation between the beads as they entered the main channel 1410.

[0452]Snapshot images from a movie of beads being separated using a T-connector are shown in FIG. 23. We developed a bead spacer to address the challenges of manipulating beads within the stacked regime (i.e., risks of clogging and loss of positional encoding at changes in channel dimension, and difficulty sorting...

example 3

Positional Encoding Device—Bead Distributing

[0458]Beads within the main channel 1410 are flowed towards the branch channels 1412, 1420. We distribute beads into branch channels by adjusting the applied pressure on the main channel upstream and downstream of each branch point 1411, 1419 and by selectively activated flow within the branch channels 1412, 1420 via the two-way selector valve 1433 such that the carrier fluid distributed each bead into its preassigned branch channel. After a first bead enters its designated branch channel, the subsequent pressure configuration and branch channel activation is determined by the branch assignment of the next bead to be distributed. If this second bead is designated for the same branch channel, the applied pressures and the two-way selector valve setting is kept the same. On the other hand, if the second bead was designated for the other branch channel, we adjust the pressures on the main channel and the flow activation of branch channels in ...

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is the National Stage of International Application No. PCT / US2020 / 012627, filed Jan. 7, 2020, which claims the benefit of and priority to U.S. Provisional Application Nos. 62 / 789,505, filed Jan. 7, 2019, 62 / 789,506 filed on Jan. 7, 2019, 62 / 810,196 filed on Feb. 25, 2019, 62 / 811,506 filed on Feb. 27, 2019, and 62 / 863,712 filed on Jun. 19, 2019, all of which are incorporated by reference in their entirety.BACKGROUND OF THE INVENTION

[0002] In biology, chemistry, and other areas it is often desirable to both create large collections of chemical compounds or products and to evaluate the characteristics, properties, performance, or utility of these products. Historically, individual products were manufactured and characterized in separate vessels. Batch type procedures have been developed and disclosed that enable production of multiple products at a time. However, due to the cost, space requirements, and physical manipulations required, there has been a longstanding desire to develop alternative methods that can produce or evaluate very large libraries of products. Approaches such as split synthesis require encoding, randomness, redundancy, and underrepresentation problems in libraries. It can be time consuming, costly, or laborious to discover the identity of the product of interest associated with a unit. Further, encoding approaches have challenges relating to cost effectiveness, scalability, speed, and accuracy.

[0003] Since the advent of PCR in 1983, the uses of customized synthetic DNA have exploded to include a rich array of applications that impact the health of millions. Among these are noninvasive prenatal tests for fetal trisomies, tests for carriers of inherited diseases, and tests that allow the selection of the cancer treatments. Synthetic DNA is also a critical input into the development of therapeutic RNAs, aptamers, and antibodies, as well as gene editing. However, orders of large numbers of DNA fragments from existing DNA suppliers result in wait-times of days and sometimes many weeks. This latency results in enormous hidden financial and opportunity costs, and fundamentally slows the pace of development of new diagnostics and therapeutics.

[0004] Therefore, there is a need for a novel microfluidic technology to enable the rapid, massively parallel, cost-efficient synthesis of nucleic acids that is able to produce thousands of high-quality nucleic acid fragments in hours. Giving scientists access to one to thousands of nucleic acid fragments, nearly on demand, stands to dramatically accelerate the pace development of new diagnostics and therapeutics, lower cost by increasing overall productivity, and open up new research paradigms based on rapid design iteration that were not previously feasible.SUMMARY OF THE INVENTION

[0005] Disclosed herein are methods and compositions relating to tracking of mobile units within a microfluidic device. In various embodiments, the tracking of mobile units is achieved by controlling or recording the position, e.g. the relative position, of the mobile units, for example as the mobile units are moving through various compartments of the microfluidic device. The tracked mobile units may be split into the channels of a microfluidic device, for example by employing a router, such as a distributor, and recombined. The order of the mobile units upon recombination may be indicative of the path each mobile unit took through the microfluidic device. Individual channels of the microfluidic device may be used to perform reactions, such as synthesis reactions. Such reactions may be performed in parallel. Reagents for each reaction may be delivered to the individual channels, for example via separate reagent delivery channels. Suitable reaction conditions, such as temperature, pressure, and flow rate may be set in the individual channels.

[0006] In a first aspect, the methods and compositions described herein relate to tracking of mobile units within a microfluidic device. The tracking may comprise moving k mobile units through a first channel of a microfluidic device in a first order; splitting the k mobile units into z branch channels; and moving the k mobile units into a second channel in a second order.

[0007] Each of the k mobile units may be mappable to one of the z branch channels based on the second order. The k mobile units may further be moved from the second channel to the first channel. The second channel may be in fluidic communication with the first channel. The steps of moving k mobile units through a first channel of a microfluidic device in a first order, splitting the k mobile units into z branch channels, and moving the k mobile units into a second channel in a second order may be repeated n times. In some embodiments, n is or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. In some embodiments, n is 2. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. The mobile units may be beads, droplets, cells, bubbles, slugs or immiscible volumes. The beads may comprise glass or silica beads, metal beads, hydrogel or polymer beads, or chemically resistant polymer beads. The microfluidic device may comprise at least i channels having a largest cross-section no greater than x times the mean cross-section of the mobile units. In some embodiments, x is or is less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.02, 1.01, or 1. In some embodiments, i is or is greater than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, or 10000. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 500, 400, 300, 250, 200, 150, 100, 90, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 micrometers. In some embodiments, j is or is greater than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, or 10000. In some embodiments, the cross-section coefficient of variation for the k mobile units is or is less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%5, 5% 4%, 3%, 2%, 1%, or less. In some embodiments, a different set of reagents is delivered to each of a subset or all of the z branch channels. The one or more sets of reagents may comprise a 2′-deoxynucleoside phosphoramidite. The first order or the second order may be predetermined. In some embodiments, z is or is more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. Each of a subset or all of the z branch channels may comprise valves at one or both ends. One or more reagent channels may be configured to deliver reagents to each of a subset or all of the z branch channels. The delivery of reagents from at least one of the one or more reagent channels may be controlled by a valve. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, or 1000000. In some embodiments, k is or is less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. In some embodiments, k is between 2 and 500.

[0008] In a second aspect, the methods and compositions described herein relate to a microfluidic device and uses thereof. The microfluidic device may comprise a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels may be configured to accept mobile units from the first channel in a first order; and a second channel in fluidic communication with the set of z branch channels, wherein the second channel may be configured to accept mobile units from the set of z branch channels in a second order. The first or the second order may be controllable. The second order may be determinative of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The microfluidic device may comprise k mobile units. The microfluidic device may comprise a router, e.g. a distributor, between the first channel and the set of z branch channels. In some embodiments, z is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. In some embodiments, k is or is less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. In some embodiments, k is between 2 and 500.

[0009] In a third aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein the synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device. The microfluidic device may further comprise i fiducial marks. The configuration of the k mobile units may depend on the relative position of j mobile units with respect to the i fiducial marks. In some embodiments, i is or is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, j is or is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, k is or is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more.

[0010] A fourth aspect of the methods and compositions described herein relates to a system comprising computer comprising a computer-readable medium; and a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein the synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device; wherein the computer is configured to record data associated with the position of the k mobile units in the computer-readable medium repeatedly.

[0011] A fifth aspect of the methods and compositions described herein relates to a system comprising a computer comprising a computer-readable medium and a microfluidic device. The microfluidic device may comprise a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; and a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order. The second order may be determinative or predictive of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The computer may be configured to record data associated with the position of the mobile units in the computer-readable medium repeatedly.

[0012] In a sixth aspect, the methods and compositions described herein relate to routing of mobile units within a microfluidic device. The method may comprise a) routing k mobile units through a first channel of a microfluidic device in a first order; b) distributing the k mobile units into z branch channels; and c) routing the k mobile units into a second channel in a second order. The routing in step a may be performed in accordance with a predetermined unit routing algorithm through the microfluidic device for at least a subset of the k mobile units. The unit routing algorithm may comprise a routing selection at at least one branch point of the microfluidic device. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels based on unit tracking information from at least one detector configured to track the movement of mobile units inside the microfluidic device. At least a subset or all of the k mobile units may be mappable to a path comprising a specific one of the z branch channels based on the second order. At least a subset of the k mobile units in step c may comprise all of the k mobile units. The first channel and the second channel may be the same. Between steps b and c, the flow direction of at least a subset of the k mobile units may be reversed. In step b, at least one unit may be routed into a first branch channel through a first branch channel end and in step c, the at least one unit may be routed out of the first branch channel through the first branch channel end. In step b, at least one unit may be routed into a first branch channel through a first branch channel end and, in step c, the at least one unit may be routed out of the first branch channel through a second branch channel end that is different than the first branch channel end. The method may further comprise routing the k mobile units from the second channel to the first channel. The second channel may be in fluidic communication with the first channel. The method may further comprise repeating steps a-c n times. n may be 2. n may be 2 to 10. n may be 10 to 100. n may be 100 to 1000. n may be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. Units may be beads. The mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. The beads comprise glass beads or polymer beads. The microfluidic device may comprise i channels having a largest cross-section x times the mean cross-section of the k mobile units. i may be 2-10000. x may be 1.05-2.0. i may be 2-100. i may be 100-1000. The microfluidic device may comprise at least i channels having a largest cross-section no greater than x times the mean cross-section of the k mobile units. The mobile units may be beads. x may be or may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or less. X may be or may be more than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or more. i may be or may be more than 2, 3, 4, 5, 10, 20, 50, 100, 1000, 5000, 10000, or more. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 200 micrometers. j may be 2 to 10000. The largest cross-section of the at least j channels may be no greater than 10 micrometers. The microfluidic device may comprise at least j channels having a largest cross-section no greater than 200 micrometers. j may be 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 5000, 10000, or more. The cross-section coefficient of variation for the k mobile units may be 1% to 20%. The cross-section coefficient of variation for the k mobile units may be 2% to 5%. The cross-section coefficient of variation for the k mobile units may be less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The method may further comprise delivering different reagents to each of the z branch channels. The reagents may comprise a 2′-deoxynucleoside phosphoramidite. The method may further comprise directing at least one mobile units into a side channel. The method may further comprising directing the at least one mobile units in the side channel to the second channel. The first order may be predetermined. The second order may be predetermined. z may be 2-10 z may be 10-100. z may be 100-1000. z may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. z may be less than 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. Each of the z branch channels may be capped by valves or unit stops on one or two ends. One or more reagent channels may be configured to deliver reagents to each of the z branch channels. Delivery of reagents from at least one of the one or more reagent channels may be controlled by a valve. Delivery of reagents from at least one of the one or more reagent channels may be controlled by application of differential pressures to selected points in the z branch channels and the reagent channels. k may be between 2 and 1000000. k may be 2-5000000. k may be 20-100. k may be 100-1000. k may be 10000-100000. k may be 100000-1000000. k may be between 2 and 500. K may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, or 20. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the second order may be verified using the at least one unit's label. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the first order may be verified using the at least one unit's label. The at least one mobile unit may comprise at least two mobile units. The labels of the at least two mobile units may be not unique.

[0013] In a seventh aspect, the methods and compositions described herein relate to a microfluidic device comprising: a) a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; and b) a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order; wherein the second order is determinative of the particular branch channel of the set of z branch channels that is configured to deliver a mobile unit in the second order. The first order or the second order may be controllable. The device may further comprise k mobile units. The device may further comprise a distributor between the first channel and the set of z branch channels. z may be between 2 and 50. z may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or more. z may be less than 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. k may be between 2 and 500. k may be between 2 and 5000000. k may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, 5000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less.

[0014] In an eight aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a synthesis history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device.

[0015] In a ninth aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a treatment history for each of the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device. The treatment history may comprise a light treatment history, a heat treatment history, an enzymatic treatment history, a cleavage treatment history, an isomerization history, an acetylation history, a synthesis history, an amplification history, or a reaction history. The microfluidic device may further comprise i fiducial marks. The configuration of the k mobile units may depend on the relative position of j mobile units with respect to the i fiducial marks. i may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. i may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. j may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. j may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or less.

[0016] In a tenth aspect, the methods and compositions described herein relate to a system comprising a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising k mobile units, wherein a different compound is associated with each of the k mobile units and wherein a treatment history of each of the different compounds associated with the k mobile units is determinable based on the configuration of the k mobile units in the microfluidic device; wherein the computer is configured to record data associated with the position of the k mobile units in the computer-readable medium repeatedly. The treatment history may comprise a light treatment history, a heat treatment history, an enzymatic treatment history, a cleavage treatment history, an isomerization history, an acetylation history, a synthesis history, an amplification history, or a reaction history.

[0017] In an eleventh aspect, the methods and compositions described herein relate to a system comprising: a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising i) a first channel in fluidic communication with a set of z branch channels, wherein the set of z branch channels is configured to accept mobile units from the first channel in a first order; ii) a second channel in fluidic communication with the set of z branch channels, wherein the second channel is configured to accept mobile units from the set of z branch channels in a second order; wherein the second order is determinative of the particular channel of the set of z branch channels that is configured to deliver a mobile unit in the second order; and wherein the computer is configured to record data associated with the position of the mobile units in the computer-readable medium repeatedly.

[0018] In a twelfth aspect, the methods and compositions described herein relate to a method of tracking, the method comprising: a) moving k mobile units through a first channel of a microfluidic device in a first order; b) routing at least a subset of the k mobile units within the microfluidic device, thereby creating a second order; c) performing a comparison of the second order to a predesignated post-routing order; and d) separating j mobile units into a correction area based on the comparison of step c by separating the j mobile units from a remainder of the at least a subset of the k mobile units; wherein each of the remainder of the at least a subset of the k mobile units is mappable to a routing path. The routing path may comprise the location of a mapped mobile unit after the routing step in step b. The routing path may comprise the location of a mapped mobile unit before the routing step in step b. The location of a mobile unit may comprise the unit's relative positional order with respect to m mapping mobile units. M may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more. m may be less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The m mapping mobile units may comprise the m closest mobile units to the mapped mobile unit along a fluidically connected path originating from the mapped mobile unit. Routing may comprise distributing into at least one branch channel of the microfluidic device. Routing may comprise merging from a plurality of branch channels of the microfluidic device. The correction area may comprise a channel of the microfluidic device. The method may further comprise merging at least one of the j mobile units with at least a subset of the remainder of the at least a subset of the k mobile units. k may be between 2 and 500. k may be between 2 and 100000. k may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be less than 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. At least one mobile unit may comprise a label. The position of the at least one mobile unit in the second order may be verified using the at least one unit's label. At least one mobile unit of the k mobile units may comprise a label. The position of the at least one mobile unit in the first order may be verified using the at least one unit's label. The at least one mobile unit may comprise at least two mobile units. The labels of the at least two mobile units may be not unique. j may be between 1 and 1000000. j may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000, or more. j may be less than 1000000, 100000, 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 7, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The method may further comprise repeating steps a-c n times. n may be 2. n may be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, 1000, or more. n may be less than 100, 750, 500, 400, 300, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. Beads may comprise glass beads or polymer beads. The comparison in step c may comprise verifying by at least one detector the position of at least one unit in the first order. The comparison in step c may comprise verifying by at least one detector the position of at least one unit in the second order. The comparison in step c may comprise counting units by at least one detector after the routing in step b is performed on one or more units, thereby generating a list of unit counts, and comparing the list of unit counts to an expected list of unit counts based on the predesignated post-routing order. The comparison in step c may comprise detecting one or more labels on one or more units by at least one detector after the routing in step b is performed on one or more units, thereby generating a list of detected unit labels, and comparing the list of detected unit labels to an expected list of unit labels based on the predesignated post-routing order.

[0019] In a thirteenth aspect, the methods and compositions described herein relate to a system comprising a) a microfluidic channel configured to carry beads in a carrier fluid; b) a detector configured to detect signals from a detection path through the microfluidic channel; and c) computer operably connected to the detector; wherein the system is calibrated to identify the signal of an isolated single bead in the microfluidic channel passing through the detection path. The system may be further calibrated to identify the signal of n adjacent beads in the microfluidic channel passing through the detection path. n may be 2 to 100. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. n may be less than 100, 90 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The system may be further calibrated to identify the signal of a gas bubble or a dust particle in the microfluidic channel passing through the detection path. The system may further comprise a router configured to route one or more beads from the microfluidic channel. The system may be configured to send a desired routing signal to the router to effectuate routing upon identification of an isolated single bead, a plurality of adjacent beads, a gas bubble or dust particle passing through the detection path. The router may comprise a distributor. The system may further comprise a bead spacer. The bead spacer may be configured to space beads flowing adjacently within the microfluidic channel. The system may further comprise a second microfluidic channel. The router may be configured to route beads into the second microfluidic channel. The router may comprise a merger.

[0020] In a fourteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising: a) a primary channel; b) a branch point; c) a first branch channel, wherein the first branch channel is fluidically connected to the primary channel through the branch point; and d) a first router configured to route units flowing in the primary channel into the first branch channel. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel and a location within the first branch channel. The device may further comprise a second branch channel, wherein the second branch channel is fluidically connected to the primary channel through the branch point. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel, a location within the first branch channel, and a location within the second branch channel. The first router may be configured to route units from the primary channel into the second branch channel by causing a pressure differential between one or more locations within the primary channel, a location within the first branch channel, and a location within the second branch channel. The device may further comprise z branch channels. The first router may be configured to route units from the primary channel into the first branch channel by causing a pressure differential between one or more locations within the primary channel and a location within the first branch channel, and pressure differentials between one or more locations within the primary channel and a location within each of the z branch channels. The router may comprise a network of fluidic outlets configured to connect to pressure controllers, such that the router is capable to regulate the fluidic pressure within channels that are connected through the branch point. The branch channels may connect to the primary channel at separate positions of the primary channel. The device may further comprise a second router configured to route units from at least one of the branch channels to the primary channel. The first router may comprise the second router. The second router may comprise a merger.

[0021] In a fifteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising a microfluidic channel holding k mobile units wherein the microfluidic device is configured to maintain the relative positional order of the k mobile units and wherein the microfluidic channel is configured to flow the k mobile units in a carrier fluid. There may be a distance greater than a minimum distance between each pair of the k mobile units measured along a path of fluidic connection. The minimum distance may be at least 1.5 times the mean diameter of the pair of the k mobile units. The minimum distance may be 2 to 10000 times the mean diameter of the pair of the k mobile units. The minimum distance may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 100, 1000, 5000, 10000, or more times the mean diameter of the pair of the k mobile units. The minimum distance may be less than 10000, 5000, 1000, 100, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, times the mean diameter of the pair of the k mobile units, or less. The width of the microfluidic channel may be at least 2 times the average diameter of the k mobile units. The width of the microfluidic channel may be at least 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000 times the average diameter of the k mobile units or more. The width of the microfluidic channel may be less than 50000, 10000, 1000, 100, 90, 80, 70, 60, 50, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2 times the average diameter of the k mobile units or less.

[0022] In a sixteenth aspect, the methods and compositions described herein relate to a method of separating beads in a microfluidic device, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second channel, wherein the first microfluidic channel and the second channel are connected by a bead spacer; b) moving a plurality of beads through the first microfluidic channel toward the bead spacer; c) passing a first bead and a second bead serially through the bead spacer into the second channel; and d) moving a carrier fluid through the second channel such that a desired length of carrier fluid is spaced between the first bead and the second bead in the second channel. Steps a-d may be repeated at least n times. n may comprise 2 to 1000000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more. n may be at most 10000000, 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The plurality of beads may comprise 2 to 1000000 beads. The plurality of beads may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more beads. The plurality of beads may comprise at most 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 21, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer beads. The desired length of carrier fluid may be 1 to 1000 times the average size of the plurality of beads. The desired length of carrier fluid may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the average size of the plurality of beads, or greater. The desired length of carrier fluid may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the average size of the plurality of beads, or less. The plurality of beads may comprise 2 to 1000000 beads. The plurality of beads may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more beads. The plurality of beads may comprise at most 10000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer beads. The first channel width may be 1 to 2 times the average diameter of the beads. The first channel width may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or 1.01 times the average diameter of the beads, or less. The first channel width may be more than 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the beads, or more. The second channel width may be 1.01 and 100 times the average diameter of the beads. The second channel width may be at least 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average diameter of the beads, or greater. The second channel width may be at most 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, or 1.01 times the average diameter of the beads, or smaller. The carrier fluid speed may be less than 50 meters / sec, 10 meters / sec, 1 meters / sec, 100 millimeters / sec, 10 millimeters / sec, 11 millimeters / sec, 0.1 millimeters / sec, or 0.01 millimeters / sec, or less. The carrier fluid speed may be at least 0.01, 0.1, 1, 10, 100 millimeters / sec, 1, 10, or 50 meters / sec, or more. The first and the second bead may be passed through the bead spacer within less than 10 sec, 1 sec, 0.1 sec, 0.01 sec, 1 msec, 0.1 msec, or 0.01 msec, or faster.

[0023] In a seventeenth aspect, the methods and compositions described herein relate to a microfluidic device comprising a microfluidic channel holding k mobile units wherein the microfluidic device is configured to maintain the relative positional order of the k mobile units and wherein the microfluidic channel is configured to flow the k mobile units in a carrier fluid. The width of the microfluidic channel may be 0.05 to 2 times the average diameter of the k mobile units measured outside of the microfluidic channel. The width of the microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.01, 1, 0.95, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.4, 0.3, 0.2, 0.1, or 0.05 times the average diameter of the k mobile units measured outside of the microfluidic channel, or smaller. The width of the microfluidic channel is more than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1, 1.01, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 1.95 times the average diameter of the k mobile units measured outside of the microfluidic channel, or greater. The device may be configured to move the k mobile units within the microfluidic channel along a moving direction of the microfluidic channel. There may be a center to center distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel of less than 2 times the average diameter of the k mobile units. The center to center distance may be 0.01 to 1.9 times the average diameter of the k mobile units. The center to center distance may be less than 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65. 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times the average diameter of the k mobile units, or less. The center to center distance may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the average diameter of the k mobile units, or greater. The device may be configured to move the k mobile units within the microfluidic channel along a moving direction of the microfluidic channel. The shortest distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65. 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The shortest distance between adjacent pairs of k mobile units within the microfluidic channel along the moving direction of the microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The maximum deviation from the average width of the microfluidic channel may be less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.10%, or less. The maximum deviation from the average width of the microfluidic channel may be more than 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more. The coefficient of variance in the diameter of the k mobile units may be less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The coefficient of variance in the diameter of the k mobile units is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0024] In an eighteenth aspect, the methods and compositions described herein relate to a microfluidic device comprising k mobile units, wherein the coefficient of variance in the diameter of the k mobile units is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. The coefficient of variance in the diameter of the k mobile units may be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more.

[0025] In a nineteenth aspect, the methods and compositions described herein relate to a method of sorting, the method comprising: a) providing k mobile units; b) introducing the k mobile units into a unit size sorter; c) separating a subset of k mobile units having sizes falling outside of a predetermined range of unit size from the remainder of the k mobile units; and d) introducing at least a subset of the remainder of the k mobile units into a microfluidic device. The upper limit of the predetermined range of unit size may be less than 1.3, 1.25, 1.2, 1.15, 1.14, 1.13, 1.12, 1.11, 1.1, 1.09, 1.08, 1.07, 1.06, 1.05, 1.03, or 1.02 times the lower limit of the predetermined range, or less. The upper limit of the predetermined range of unit size may be more than 1.02, 1.03, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.2, 1.25, or 1.3 times the lower limit of the predetermined range, or more.

[0026] In a twentieth aspect, the methods and compositions described herein relate to a method of separating units in a microfluidic device, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second channel, wherein the first microfluidic channel and the second channel are connected by a unit spacer; b) moving a plurality of units through the first microfluidic channel toward the unit spacer; c) passing a first unit and a second unit serially through the unit spacer into the second channel; and d) moving a carrier fluid through the second channel such that a desired length of carrier fluid is spaced between the first unit and the second unit in the second channel. The steps a-d may be repeated at least n times. n may be 2 to 1000000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, or more. n may be at most 10000000, 1000000, 100000, 10000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The plurality of units may comprise 2 to 1000000 units. The plurality of units may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000 or more units. The plurality of units may comprise at most 1000000, 100000, 100000, 5000, 1000, 500, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer units. The desired length of carrier fluid may be 1 to 1000 times the average size of the plurality of units. The desired length of carrier fluid may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the average size of the plurality of units, or greater. The desired length of carrier fluid may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the average size of the plurality of units, or smaller. The first channel width may be 1.1 to 2 times the average diameter of the units. The first channel width may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 times the average diameter of the units, or smaller. The first channel width may be more than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the units, or greater. The second channel width may be 1.05 to 100 times the average diameter of the units. The second channel width may be at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average diameter of the units, or greater. The second channel width may be at most 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.05 times the average diameter of the units, or smaller. The carrier fluid speed may be at least 0.01, 0.1, 1, 10, 100 millimeters / sec, 1, 10, or 50 meters / sec, or faster. The carrier fluid speed may be less than 50 meters / sec, 10 meters / sec, 1 meters / sec, 100 millimeters / sec, 10 millimeters / sec, 11 millimeters / sec, 0.1 millimeters / sec, or 0.01 millimeters / sec, or slower. The first and the second unit may be passed through the unit spacer within 0.01 msec to 10 sec. The first and the second unit may be passed through the unit spacer within less than 10 sec, 1 sec, 0.1 sec, 0.01 sec, 1 msec, 0.1 msec, 0.01 msec, or faster. The microfluidic device may be configured to maintain the relative positional order of the plurality of units. The plurality of units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. Beads may comprise glass beads or polymer beads.

[0027] In a twenty first aspect, the methods and compositions described herein relate to a system comprising: a) a computer comprising a computer-readable medium; and b) a microfluidic device comprising r routers and c microfluidic channels in fluidic connectivity, wherein the r routers are configured to route k mobile units through at least a subset of the c microfluidic channels; and c) d detectors operably connected to the computer, wherein the detectors are configured to detect signals from detection paths through the at least c microfluidic channels or the at least r routers; wherein the computer is configured to record data associated with detected signals from the at least d detectors in the computer-readable medium repeatedly and to generate routing paths for at least a subset of the k mobile units. c may be 2 to 1000. c may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. c may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less. d may be 2 to 1000. d may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. d may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. r may be 2 to 1000. r may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. r may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. k may be 2 to 1000000. k may be at least at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The system may be further configured to route at least j units of the k mobile units to a first channel of the c microfluidic channels n times. n may be 2 to 1000. n may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 32, 40, 48, 50, 60, 64, 70, 72, 80, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. n may be at most 10000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 96, 90, 80, 72, 70, 64, 60, 50, 48, 40, 32, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. j may be 2 to 5000000. j may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, 5000000, or more. j may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The k mobile units may be selected from the group consisting of beads, droplets, cells, bubbles, slugs and immiscible volumes. The c routers may comprise one or more distributors, mergers, or spacers. The routing path may comprise the location of a mapped mobile unit downstream of a router. The routing path may comprise the location of a mapped mobile unit upstream of a router. The location of a mobile unit may comprise the unit's relative positional order with respect to m mapping mobile units. m may be 1 to 100. m may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more. m may be at most 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. The m mapping mobile units may comprise the m closest mobile units to the mapped mobile unit along a fluidically connected path originating from the mapped mobile unit. The r routers may be configured to route mobile units in accordance with a predetermined unit routing algorithm through the microfluidic device. The computer may be configured to perform a comparison between a first post-routing order for the at least a subset of the k mobile units after a routing event by at least one of the r routers and a predesignated post-routing order. The computer may be configured to generate routing paths for i of the at least a subset of the k mobile units based on the comparison. The r routers may be configured to route i mobile units in accordance with the routing paths for the i mobile units. i may be 2 to 1000000. i may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. i may be at most 5000000, 1000000, 500000, 100000, 50000, 10000, 1000, 500, 100, 50, 30, 20, or less. The r routers may be configured to separate j mobile units from a remainder of the at least a subset of the k mobile units into a correction area based on the comparison. The r routers may be configured to route mobile through the microfluidic device randomly.

[0028] In a twenty second aspect, the methods and compositions described herein relate to a method of tracking, the method comprising: a) providing a microfluidic device comprising a first microfluidic channel and a second microfluidic channel in fluidic connection with the first microfluidic channel; and b) routing k mobile units through the first microfluidic channel into the second microfluidic channel in ordered flow. The first microfluidic channel and the second microfluidic channel may be the same. The first microfluidic channel and the second microfluidic channel may be connected by a union, unit spacer, distributor, or merger. The microfluidic device may further comprise a third microfluidic channel. The method may further comprise routing the plurality of mobile units through the second microfluidic channel into the third microfluidic channel in ordered flow. The second microfluidic channel and the third microfluidic channel may be the same. The first microfluidic channel and the third microfluidic channel may be the same. The second microfluidic channel and the third microfluidic channel may be connected by a union, unit spacer, distributor, or merger. The width of the first microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the first microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the first microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the second microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the second microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the second microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller. The width of the third microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the third microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the third microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the first microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the first microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the first microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller. The width of the second microfluidic channel may be 0.01 to 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel. The width of the second microfluidic channel may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or smaller. The width of the second microfluidic channel may be greater than 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 times the average diameter of the k mobile units as measured outside of the microfluidic channel, or greater. The width of the third microfluidic channel may be 1.05 to 100 times the average diameter of the units. The width of the third microfluidic channel may be greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times the average diameter of the units, or greater. The width of the third microfluidic channel may be less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05 times the average diameter of the units, or smaller.

[0029] In a twenty third aspect, the methods and compositions described herein relate to a method of synthesizing oligomers associated with mobile units, the method comprising: a) routing k mobile units through a first channel of a microfluidic device in a first order; b) distributing at least a subset of the k mobile units into at least z branch channels; and c) routing the at least a subset of the k mobile units into a second channel in a second order; wherein at least a subset of the k mobile units are functionalized with a group suitable to synthesize an oligomer; wherein at least a subset of the k mobile units are mappable to a path comprising a specific one of the z branch channels; wherein at least a subset of the k mobile units are subjected to reaction conditions comprising conditions for a step of a synthesis reaction inside the z branch channels; and wherein steps a-c are repeated for n cycles. In some embodiments, the synthesis reaction comprises a nucleic acid synthesis reaction or a peptide synthesis reaction. In some embodiments, the reaction conditions comprise an enzyme. In some embodiments, the enzyme is selected from a terminal deoxynucleotidyl transferase, a thermostable DNA polymerase, a DNA polymerase theta, a Poly(A) polymerase, and a DNA polymerase encoded by a variant of the 9°N DNA Polymerase gene from Thermococcus species 9°N-7. In some embodiments, the variant of the 9°N DNA Polymerase gene comprises the 9°N (D141A / E143A / A485L) DNA Polymerase gene or the 9°N (E143D) DNA Polymerase gene. In some embodiments, the enzyme is conjugated to a nucleotide or a nucleotide analog. In some embodiments, the reaction conditions comprise a nucleotide or a nucleotide analog. In some embodiments, at least a subset of the k mobile units are functionalized with an initiator nucleic acid or a nascent oligonucleotide. In some embodiments, the nucleic acid synthesis reaction is a template independent nucleic acid synthesis reaction. In some embodiments, the method further comprises performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal nucleotide of initiator nucleic acids or nascent oligonucleotides associated with at least a subset of the k mobile units and a new nucleotide or nucleotide analog in the presence of a transferase enzyme. In some embodiments, the new nucleotide or nucleotide analog comprises a blocking moiety. In some embodiments, the method further comprises performing a deblocking reaction thereby removing the blocking moiety from the newly incorporated nucleotide or nucleotide analog. In some embodiments, the method further comprises one or more steps selected from the group consisting of a washing step, a modification step, a cleaving step, and a capping step. In some embodiments, two or more of the steps selected from the group consisting of the coupling reaction, the deblocking reaction, the washing step, the modification step, the cleaving step, and the capping step are performed in different cycles. In some embodiments, the oligomers are oligonucleotides and wherein the method further comprises assembling the oligonucleotides into genes. In some embodiments, assembling of the oligonucleotides into genes is performed using one or more method selected from the group consisting of polymerase-cycling assembly, enzymatic gene assembly, annealing and ligation reaction, shotgun ligation, shotgun ligation and co-ligation, gene synthesis via one strand, template directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron technology, Sloning building block technology, Golden Gate assembly, Dual-Asymmetric (DA) PCR, Overlap Extension (OE) Asymmetric PCR, Thermodynamically-Balanced Inside Out (TBIO), Two-Step (DA+OE), Polymerase Assembly Multiplexing (PAM), One-Step Simplified Gene Synthesis, Single Molecule PCR, TopDown Real-Time Gene Synthesis, Two-step Ligation and PCR, Brick-based assembly, Sequence- and Ligation-Independent Cloning (SLIC), Transformation-associated Recombination, Biobrick assembly, PCR-based two-step DNA synthesis (PTDS), successive PCR method. In some embodiments, the reaction conditions comprise one or more of reagents selected from the group consisting of an amino acid, a dipeptide, a polypeptide, and a carbodiimide. In some embodiments, the method further comprises performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal end of nascent peptides associated with at least a subset of the k mobile units and a new amino acid, dipeptide or polypeptide. In some embodiments, the method further comprises performing one or more step selected from the group consisting of a capping step, a washing step, and a deprotecting step. In some embodiments, two or more of the steps selected from the group consisting of the coupling reaction, capping step, washing step and the deprotecting step are performed in different cycles. In some embodiments, the same z branch channels are used in at least two of the n cycles. In some embodiments, n is 2. In some embodiments, n is 2 to 10. In some embodiments, n is 10 to 100. In some embodiments, n is 100 to 200. In some embodiments, n is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 750, or 1000. In some embodiments, n is at most 1000, 750, 500, 400, 300, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. n may fall within a range bounded by any of the foregoing values, e.g. 100-200, 200-300, 300-400, 400-500, 500-1000. In some embodiments, z is 2-100. In some embodiments, z is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. In some embodiments, z is at most 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2. z may fall within a range that is bounded by any of the foregoing values, e.g. 2-100, 2-16, 4-20, 2-24, etc. In some embodiments, k is at most 1000000000000, 100000000000, 10000000000, 1000000000, 100000000, 10000000, 1000000, 100000, 10000, 1000, 500, 100, or less. In some embodiments, k is between 20-500. In some embodiments, k is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 10000, 50000, 100000, 500000, 1000000, or more. k may fall within a range that is bounded by any of the foregoing values, e.g. 2-50000, 10-50, 20-500 etc. In some embodiments, the mobile units are selected from the group consisting of beads, droplets, cells, bubbles, slugs, immiscible volumes, glass beads, polymer beads, cross-linked beads, cross-linked polymer beads, divinylbenzene cross-linked polymer beads, and divinylbenzene cross-linked polystyrene beads. In some embodiments, the first order is different in at least two of the n cycles. In some embodiments, the second order is different in at least two of the n cycles. In some embodiments, the first channel is the same as the second channel.

[0030] In a twenty fourth aspect of the methods and compositions described herein relate to a composition comprising n cross-linked beads wherein the coefficient of variation for bead diameter is less than 20%. In some embodiments, the coefficient of variation for bead diameter is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. In some embodiments, the mean diameter of the beads is between 0.1-500 μm. In some embodiments, the mean diameter of the beads is between 10-50 μm. In some embodiments, the mean diameter of the beads is between 20-150 μm. In some embodiments, the mean diameter of the beads is at least 0.1 μm, 1 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 150 μm 200 μm, 300 μm, 400 μm, 500 μm, or greater. In some embodiments, the mean diameter of the beads is at most 500 μm, 400 μm, 300 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 75 μm, 70 μm, 60 μm, 50 μm, 40 m, 30 μm, 20 μm, or smaller. The mean diameter of the beads may fall within a range bounded by any of the foregoing values, e.g. 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 20-50 μm, 80-90 μm, 90-100 μm, 0.1-400 μm, etc. In some embodiments, the beads are functionalized with a group suitable for oligomer synthesis. In some embodiments, the beads are functionalized with one or more groups selected from the group consisting of amine, hydroxyl, chloromethyl, aminomethyl, benzhydrodrylamino, silane, alkylsilane, and carboxyl groups. In some embodiments, the beads are cross-linked at a molar cross-linker ratio of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more. In some embodiments, the beads are cross-linked at a molar cross-linker ratio of at most 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or less. The beads may be cross-linked at a molar cross-linker ratio that falls within any range bounded by the foregoing values, e.g. 20-40%, 15-50%, 25-45% etc. In some embodiments, the beads are cross-linked using one or more cross-linkers selected from the group consisting of divinylbenzene, glutaraldehyde, formaldehyde, an epoxy compound, dialdehyde, and dichloroethane. In some embodiments, the beads are cross-linked using radiation or oxidation. In some embodiments, the variation in bead diameter suspended in organic solvent versus aqueous solvent is less than 80%, 30%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less. In some embodiments, the variation in bead diameter suspended in organic solvent versus aqueous solvent is greater than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 30%, 50%, or more. Variation in bead diameter suspended in organic solvent versus aqueous solvent may fall within a range bounded by any of the foregoing values, e.g. 0.5-2%, 1-5%, 2-10%, 1-50% etc. In some embodiments, the organic solvent is selected from the group consisting of toluene, acetonitrile, toluene, dichloromethane, tetrahydrofuran (THF), pyridine, N-methyl pyrrolidinone (NMP), 2,6-lutidine, carbon disulfide, 1,2-dichloroethane, 1,1-dichloroethane, chloroform, dimethylformamide, dimethylacetamide, dimethylsulfoxide (DMSO), ethylene carbonate, 1,4-dioxane, DME (1,2-dimethoxyethane), nitromethane, methyl tert-butyl ether, methyl ethyl ketone (butanone), and dichloromethane. In some embodiments, n is at least 500, 1000, 10000, 100000, 1000000, 5000000, 10000000, 100000000, 1000000000, 10000000000, 100000000000, 1000000000000 or more. In some embodiments, n is at most 1000000000000, 100000000000, 10000000000, 1000000000, 100000000, 10000000, 1000000, 100000, 10000, 1000, or less. n may fall within a range bounded by any of the foregoing values, e.g., 500-10000, 1000-10000000000, 10000-1000000000000 etc. In various embodiments, bead compositions described herein are contained in a microfluidic device.

[0031] In a twenty fifth aspect the methods and compositions described herein relate to a system comprising: a) a microfluidic device comprising i delivery channels each in fluidic communication with a different set of z branch channels, wherein each of the sets of z branch channels is configured to accept a plurality of mobile units in a first order from one of the i delivery channels through a branch point; b) one or more routers configured to route mobile units into one of the z branch points at the first branch point; and c) a controller configured to control the one or more routers to route mobile units into one of the z branch points at the first branch point; wherein the first order is determinative of the particular branch channel of the set of z branch channels into which the controller is configured to control the one or more routers to route a specific mobile unit. The system may further comprise j outlet channels in fluidic communication with some or all of the set of z branch channels, wherein the outlet channels are configured to accept mobile units from the branch channels in a second order. In some embodiments, the second order is determinative of the particular branch channel of the set of z branch channels that is configured to deliver a specific mobile unit into one of the outlet channels. In some embodiments, the delivery channels and the outlet channels are the same. In some embodiments, at least a subset of the one or more routers comprise one or more microfluidic structures configured to generate a vortex. In some embodiments, the microfluidic structures configured to generate a vortex are positioned a distance from the branch point that is at least 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 1000 m, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or greater. In some embodiments, the microfluidic structures configured to generate a vortex are positioned a distance from the branch point that is at most 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, or smaller. The microfluidic structures configured to generate a vortex may be positioned a distance from the branch point that falls within a range bounded by any of the foregoing values, e.g. 100 μm-2.3 mm, 200 μm-450 μm, 150 μm-750 μm, etc. In some embodiments, at least a subset of the one or more routers comprise a thermal bubble forming apparatus. In some embodiments, z is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more. In some embodiments, z is at most 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2. z may fall within a range bounded by any of the foregoing values, e.g. 2-50, 4-20, 3-10, etc. In some embodiments, i is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000 or more. In some embodiments, i is at most 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. i may fall within a range bounded by any of the foregoing values, e.g. 2-30, 3-20, 10-500, etc In some embodiments, j is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000 or more. In some embodiments, j is at most 1000, 500, 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. j may fall within a range bounded by any of the foregoing values, e.g. 1-10, 5-20, 10-100, etc In some embodiments, i equals j.

[0032] In a twenty sixth aspect, the methods and compositions described herein relate to a method of DNA assembly, comprising: disposing a solid phase support column in a chamber of a microfluidics circuit; performing an enzymatic reaction in a channel of the microfluidics circuit to produce a reaction mixture, the channel fluidly coupled to the chamber; and flowing the reaction mixture over the solid phase support column in the chamber to capture assembled oligos. In some embodiments, the method further comprises flowing a wash mixture over the solid phase support column to remove remaining portions of the reaction mixture. In some embodiments, the method further comprises flowing an elution fluid over the solid phase support column to elute the assembled plurality of oligonucleotides.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] These and other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures, wherein:

[0034] FIG. 1 provides an illustrative example of a microfluidic device comprising a first primary channel having a plurality of ordered mobile units, such as beads. A router, e.g. a distributor, (triangle) at the connection of the first channels with two branch channels can serve to direct each of the mobile units into one of the two branch channels. Valves in the two branch channels may be configured to control entry and exit of the mobile units. Reagents may be delivered to the two branch channels via reagent delivery channels. Delivery of reagents may be controlled with a valve. This configuration can be representative of one of many iterative steps a plurality of beads may undergo through the microfluidic device. The circles with numbers depict units with unit ID numbers; the rectangles depict valves; and the triangles depict routers, e.g. distributors.

[0035] FIG. 2 provides an illustrative example of a microfluidic device. Mobile units 1-6 from a first channel are being directed deterministically into one of two branch channels by using a router, e.g. a distributor. Beads 7-9 are arranged in the first channel, soon to enter the router. The router is programmed to deliver beads 7-9 the positions indicated by the hashed-circles. Once the mobile units are distributed into the branch channels, reagents, such as synthesis reagents may be circulated through the two branch channels that will be holding the mobile units.

[0036] FIG. 3 provides an illustrative example of a snapshot of tracked circulating of mobile units through split channels of a microfluidic device. The order of the mobile units in the channel as the mobile units are about to start a new round is different than the order shown in FIG. 1. The order of the mobile units as they are recirculated back to the first channel may be set in a deterministic manner. The position or relative position of specific mobile units may be known. In this illustrative example, the mobile units are being prepared to be distributed again into the branch channels that may be set to host a pre-assigned sequence of chemistries.

[0037] FIG. 4 provides an illustrative example of a microfluidic device wherein mobile units are split into four branch channels passing through two sets of successive routers, e.g. distributors. A device configuration with four branch channels may be used to synthesize nucleic acids in or on the mobile units by successive circulation of the mobile units through the branch channels. Dedicated reagent delivery channels may each provide one of four building blocks for nucleic acid synthesis.

[0038] FIG. 5 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. Valves in each of the four channels may control exit and entry of the mobile units and create a reaction chamber for a reaction cycle comprising chemical modification of the units when closed. Units released from one or more of the reagent chambers may be merged with the units released from another reaction chamber at successive branch points, resulting in combination of the units in the four channels into two channels.

[0039] FIG. 6 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. A detector in the two channels after the first router may interrogate the units as they pass through the channels. The data may be sent to a computer for storage and image processing.

[0040] FIG. 7 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. In this example, the units may be distributed into a reaction cluster comprising four reaction chambers with three consecutive valves: a first valve, a middle valve, and a last valve. These valves may form two reaction chambers in each channel, resulting in eight total reaction chambers in the reaction cluster.

[0041] FIG. 8 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. After the units undergo a reaction cycle in some or all of the reaction chambers, the units may be re-combined by flowing them through channels that merge, according to an algorithm or randomly. In this example, the two middle channels merge with each other first, before merging with the left (top) and the right (bottom) channels.

[0042] FIG. 9 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of routers, e.g. distributors. In this example, the units are distributed into different channels with varying numbers of reaction chambers.

[0043] FIG. 10 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through two sets of successive routers, e.g. distributors. The reaction chambers may include additional features not shown, as indicated by the broken lines in the channel.

[0044] FIG. 11 provides an illustrative example of a microfluidic device with two consecutive reaction clusters.

[0045] FIG. 12 provides an illustrative example of a microfluidic device with two consecutive reaction clusters. In this example, the reaction chambers may include additional features not shown, as indicated by the broken lines in the channel.

[0046] FIG. 13 provides an illustrative example of a microfluidic device with a plurality of reaction zones. Units distributed into the different reaction zones may undergo the same reaction, different reactions, or no reaction. Reactions may occur simultaneously, consecutively, or at different times.

[0047] FIG. 14 provides an illustrative example of a microfluidic device with unit spacers wherein mobile units are distributed into two branch channels. The unit spacer(s), unit stop(s), and / or the pressure controller(s) and / or regulator(s) may be used to space and distribute units into branch channels and merge units from the branch channels.

[0048] FIG. 15 provides an illustrative example of a microfluidic device wherein mobile units are distributed into four branch channels passing through a spacer and two sets of successive routers, e.g. distributors.

[0049] FIG. 16A provides an illustrative example of a detection system. FIG. 16B provides a photograph of a unit doublet traveling through an optical detection system.

[0050] FIG. 17A-C provide examples of the signal generated by unit singlets (17A), unit doublets (17B), and unit singlets, doublets and multiples (17C) as they pass through the optical detection system of FIG. 16B configured in accordance with the schematics shown in FIG. 16A.

[0051] FIG. 18A-B provides examples of signals generated by single units (18A) and bubbles (18B).

[0052] FIG. 19 provides an illustrative example of a set-up for bead manipulation.

[0053] FIG. 20 provides an illustrative example for a bead mixing mechanism with reagents.

[0054] FIG. 21 provides an image of a double T-junction branch point.

[0055] FIG. 22A-D provides images of a unit stop (A), a unit spacer (B), a unit spacer with polished capillaries inserted (C) and a cross channel unit spacer (D).

[0056] FIG. 23A-D provides snapshots from a movie of beads being separated by a unit spacer.

[0057] FIG. 24A-D provides pictures of (A) a unit stop constructed from a LabSmith union connector, (B) a close up image of the capillary, tubing, and wires in the unit stop of panel (A), (C)-(D) close up images of a wire inserted into a capillary for use as a unit stop with fitting removed, showing wire.

[0058] FIG. 25 provides an image of an exemplary positional encoding device.

[0059] FIG. 26 provides a diagram illustrating exemplary error correction methods and devices in accordance with various embodiments of the invention.

[0060] FIG. 27 provides an illustrative example of a microfluidic device and system comprising a multichannel pressure / flow controller (OB1 Mk3, Elveflow), fluid reservoirs, fluid flow sensors, and automated 2-way valves (LabSmith).

[0061] FIG. 28 depicts a close-up image of an illustrative microfluidic device and system focusing on the pressure controller and reservoirs. Shown are tubing from pressure controller outputs to reservoir caps through a filter and / or a liquid stop 2801, which are pneumatic lines that are configured to pressurize the reservoirs; and 360 um fused silica capillary fluid lines leading from top of reservoirs 2802.

[0062] FIG. 29 depicts an illustrative fluidic breadboard with flow sensors and automated valves. Input fluid lines pass through the flow controllers to the two-way valves. Two-way valves route flow to different parts of the fluidic network. The left valve directs flow to the “top” or “bottom” of a main transport channel (the second channel in FIG. 25 described in further detail elsewhere herein.

[0063] FIG. 30 provides a close-up image of a microfluidic fluid flow sensor (MFS, Elveflow). Top cable is configured to deliver fluid flow data to the multichannel pressure / flow controller depicted in FIG. 27. With the use of the flow sensor, the multichannel pressure / flow controller can be used to perform closed loop control of fluid speed by dynamically adjusting the applied pressure.

[0064] FIG. 31A-F provides diagrams of differential pressure for distributing units in a single T junction.

[0065] FIG. 32A-E provides diagrams of differential pressure for distributing units in a double T junction.

[0066] FIG. 33 provides a close-up image of a cross-flow routing junction. Glass capillaries are inserted into 3D printed routing device. Units, e.g. beads, spaced 40 μm from each other enter from left and a cross-flow current from the vertically oriented capillaries deflect the units into the upper or lower bifurcation.

[0067] FIG. 34A-B provide a schematic (34A) of electroosmotic flow and a close up of an electroosmotic pump (34B). Voltage across two porous electrodes drives electroosmotic flow through a nanoporous pump medium. Electrical leads drive fluidic flow through connected glass capillaries.

[0068] FIG. 35A-C provide close up images on a unit trap (35A), a unit spacer (35B), and a laser detector (35C). FIG. 35A illustrates an exemplary 3D printed unit trap: A disk shaped element with slit in channel is configured to allow fluid flow while preventing units from passing. FIG. 35B illustrates an exemplary unit spacer that is configured to introduce spaces between stacked units. FIG. 35C illustrates an exemplary laser unit detector: 40 μm units, such as beads may be detected by laser as they move left to right in channel.

[0069] FIG. 36 depicts swelling response of divinylbenzene-cross-linked polystyrene (PS) beads in organic solvent versus percent divinylbenzene (DVB) cross-linking agent.

[0070] FIG. 37A-F depict images and schematics of an example device portion for spacing and re-stacking beads without permanent jams. FIG. 37A is a plan view of the example device; FIG. 37B is an elevation view of a schematic of the example device; FIG. 37C is a snapshot of a video of a region of view about a first spacer in the example device;

[0071] FIG. 37D is an image of a detector region of the example device; FIG. 37E is an image of signals produced as beads pass the detector; and FIG. 37F is a snapshot of a video of a region of view about a second spacer in the example device, which when operated in reverse removes or reduces excess fluid between beads and returns them to a more packed configuration.

[0072] FIG. 38 depicts images of an example device portion for steering beads using an in-line spacer element coupled to a Y-channel. Additional, fluidic connections at the Y-channel junction deliver a volume of fluid into the Y-junction, causing beads to be deflected and move into the opposing leg of the bifurcation.

[0073] FIG. 39 depicts an exemplary illustration of a router configured to distribute beads into a plurality of branch channels and / or merge beads from a plurality of branch channels.

[0074] FIG. 40 depicts an exemplary illustration of a router configured to route units into a plurality of branch channels using bubbles generated by a microactuator.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Briefly, and as described in more detail below, described herein are methods and compositions relating to tracking of mobile units within microfluidic devices. Mobile units may be tracked by controlling or recording the relative positioning of the mobile units within the microfluidic device. The tracked mobile units may be distributed into the channels of a microfluidic device, for example by employing a router, such as a distributor, and recombined. The order in which mobile units move through the microfluidic device as they are split into and are recombined from various compartments of the microfluidic device may be controlled and / or recorded. The order or relative position of the mobile units upon recombination may be used to determine the path each mobile unit took through the microfluidic device. Individual channels of the microfluidic device may be used to perform reactions, such as synthesis reactions, e.g. nucleic acid synthesis reactions. Such reactions may be performed in parallel. Reagents for each reaction may be delivered to the individual channels, for example via separate reagent delivery channels. Suitable reaction conditions, such as temperature, pressure, and flow rate may be set in the individual channels. The mobile units may comprise beads such as glass beads, polymer beads, or chemically resistant polymer beads. Synthesis reactions may be performed on a nascent chain on the beads. The mobile units may or may not carry labels or barcodes. The terms ‘label’ and ‘barcode’ shall be used interchangeably herein.

[0076] Provided herein are methods of positionally tracking and moving units within a microfluidic device. The units may be loaded into a microfluidic device. In relation to jamming, clogging, aggregating, and / or keystoning of beads (or other units) at a capillary opening into the microfluidic device, embodiments of the system(s) and method(s) described can implement structures that facilitate loading of beads (or other units) into a capillary in a desired manner.

[0077] In a first embodiment, a first end of a capillary is inserted into a reservoir containing the beads (or other units) in solution, and applying positive pressure to the reservoir to drive beads (or other units), with the solution, into the capillary in a desired manner. Positive pressure can be applied by way of a pump coupled to the reservoir, where the applied pressure can be configured in a manner that controls the rate at which fluid and beads (or other units) are driven into the end of the capillary interfacing with the reservoir.

[0078] Additionally or alternatively, in a second embodiment, a first end of a capillary is inserted into a reservoir containing the beads (or other units) in solution, and a negative pressure (e.g., vacuum) is applied to another region of the capillary (e.g., a second end) to deliver beads (or other units), with the solution, into the capillary in a desired manner. Negative pressure can be applied in a manner that controls the rate at which fluid and beads (or other units) are driven into the end of the capillary interfacing with the reservoir.

[0079] In any of the embodiments of loading described herein, loading beads (or other units) into a capillary can be supplemented with agitation of the vessel or the solution containing the beads (or other units) or by adjusting concentration of the units in solution to modulate bead / unit throughput, and / or to minimize simultaneous bead arrival at the capillary opening(s). For instance, the vessel containing the beads may be affixed to a device that provides any motion including vibration, rocking, shaking, rotation, randomly or periodically. Such motion may be achieved through use of a piezoelectric device, an ultrasound device, or a mechanical mechanism. Additionally or alternatively, fluid containing the beads may be circulated or agitated inside an otherwise stationary vessel using ultrasound, stirrers, stir-bars, paddles, propellers, or additional fluid connections delivering jets of fluids. Additionally or alternatively, a dilution reagent that affects concentration and / or viscosity of the solution can be used to flow beads or units through the device or capillary.

[0080] Additionally or alternatively, in any of the embodiments of loading described herein, the capillary and / or channels fluidly coupled to the capillary upstream of the capillary can be configured with various geometries that reduce the likelihood that multiple beads arrive at the capillary opening simultaneously or in another undesired manner. Such geometries can include changes (e.g., step changes, gradual changes) channel width or diameter, or in wall width or diameter along a downstream-to-upstream direction, which can yield a configuration where the beads or other units are transmitted from the reservoir into the capillary one-by-one. Beads or other units may then be rapidly accelerated into the capillary due to increasing flow rates with smaller cross-sectional area along the flow path into the capillary.

[0081] Additionally or alternatively, in any of the embodiments of loading described above, the system(s) and / or method(s) described herein can be configured to apply and / or take advantage of forces exerted upon the beads or other units opposite to the unit loading direction, thereby controlling unit loading into the capillary. For instance, in embodiments, one or more of: gravitational forces, magnetic forces, electrophoretic forces, dielectrophoretic forces, fluid forces (e.g., using ultrasound, vibration, agitation, or a sheathing stream surrounding a core stream of fluid), and other forces are used alone or in combination with any of the above-described embodiments. Such applications may be used to mitigate or eliminate jamming or a likelihood of simultaneous bead / unit arrival at an opening of the capillary.

[0082] In another such embodiment of a configuration of bead loading using opposing forces, a loading capillary is oriented downward (e.g., parallel to gravitational forces), such that the beads or other units have a natural propensity to flow downward into the capillary due to gravitational forces. Gravity can thus carry the beads or other units, with solution, into the capillary, while an opposing force (e.g., applied negative pressure in an “upward” direction) counters the tendency for units to form aggregates at the opening into the capillary.

[0083] In another such embodiment of a configuration of bead loading using opposing forces, a loading capillary is oriented upward (e.g., parallel to gravitational forces), such that the movement of beads into the capillary is countered by gravitational forces. Suction of units, with solution, into the capillary, while gravity serves as an opposing force counters the tendency for units to form aggregates at the opening into the capillary.

[0084] In various embodiments the opposing forces already described can be applied intermittently, so as to counter or disrupt aggregates that may have formed at the opening into the capilliary. In the case of gravity, which cannot be intermittently applied, the suction (e.g. negative pressure applied to the fluid in the capillary causing beads and fluid to be drawn into the capillary) or positive pressure (e.g. positive pressure inside the vessel that causes beads and fluid to move into the opening of the capillary) may be intermittently reduced or stopped, so counter or disrupt aggregates that may have formed at the opening of the capillary.

[0085] In various embodiments, channels or other fluidic structures fluidly coupled to the capillary can include grooves or similar structures that facilitate formation of rows of units prior to entering the opening of the capillary mitigating likelihood of aggregation at the opening. In variations, however, the capillary may not be oriented upward or downward (e.g., parallel to gravitational forces) and can alternatively be oriented at any angle between vertically upward or vertically downward.

[0086] In various embodiments, the systems described herein comprise elements that apply opposing magnetic forces (e.g., for magnetic units) and / or opposing electric forces (e.g., for charged units) or other suitable types of forces described herein or known in the art. Such forces can be used to control flow of beads or other units into a loading channel, e.g. a loading capillary. For instance, adjustment of a net force applied to each unit (e.g. bead) as it enters a capillary, such as through opposing electromagnets or permanent magnets that are placed at an adequate distance relative to the capillary, can be used to control position, velocity, and / or acceleration of unit(s) (e.g. beads) into and within the capillary in a manner that prevents jamming or other undesired transmission.

[0087] Additionally or alternatively, in another such embodiment, a Bernoulli force applied across a second end of the capillary away from a first end of the capillary can be used to generate a pressure reduction that promotes transmission of the bead(s) or other units into the capillary in a desired manner. The system can apply a Bernoulli force through structures providing crossflow of fluid across the second end of the capillary.

[0088] In various embodiments, unit loading channels are oriented directly parallel or directly opposite forces applied to units in solution prior to loading, or at any other suitable angle. Forces applied to units in pre-loading solution may be adjusted such that the component of the force along the direction of the flow has a suitable absolute or relative value (e.g. relative to the pressure differential for flow into the loading channel) that can mitigate or eliminate aggregation at the entrance of the loading channel.

[0089] Provided herein are also methods of spacing or ejecting units within a microfluidic device. Provided herein are methods of steering or distributing units within a microfluidic device. Provided herein are methods of trapping or holding units within a microfluidic device. Provided herein are methods of tracking units within a microfluidic device. Provided herein are methods of dispensing units within a microfluidic device.

[0090] Provided further herein are methods to prototype fluidic components and networks including micromachining, soft lithography with polydimethylsiloxane (PDMS), 3D printing, and photolithography. In various embodiments, such methods are used to perform fundamental operations for microfluidic devices and systems described herein (e.g. a desktop synthesizer), including without limitation operations such as moving, stacking, spacing, steering, and counting units, e.g. beads. In various embodiments, stacking of units is achieved by tightly packing units in the microfluidic devices described herein. In some embodiments, stacking is used to allow for efficient application of reaction conditions or treatments (e.g. those aimed to achieve desired chemical reactions) in small reagent volumes. In some embodiments, spacing of units during various routing operations is used to enable deterministic deflection of individual units, e.g. beads. In various embodiments, a method for optical detection is used in order to reliably detect and count units at one or multiple points within the device, allowing for accurate tracking of units some or all times, even in the event that a unit is incorrectly steered.

[0091] In further embodiments, use of highly spherical and uniformly sized units allows for thousands of such to be driven as a one-dimensional (1D) array through narrow channels, for example by application of fluidic pressures. In some embodiments, such units in a 1D array hundreds of times in succession are driven through the microfluidic devices described herein without jamming, clogging, or fragmenting.

[0092] It must be noted that, as used in the specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearly dictates otherwise.

[0093] Using phosphoramidite DNA synthesis chemistry molecules can be synthesized on the surface of a solid support substrate in a step-by-step reaction proceeding, generally, in the 3′ to 5′ direction and consisting of (1) a detritylation step to remove a protecting group from the previously added nucleoside (this prevents more than one nucleoside from being added per cycle), (2) a coupling of the next nucleoside to the growing DNA oligomer, (3) oxidation to convert the phosphite triester intermediate into a more stable phosphate triester, (4) irreversibly capping any unreacted 3′ hydroxyls groups. Without being bound by theory, capping unreacted 3′ hydroxyl groups can help prevent synthesized sequences having a deletion relative to preselected nucleic acid sequences by avoiding continued polymerization from such 3′ hydroxyl groups in subsequent cycles. The cycle can be repeated to add the next base. Solid supports may comprise a variety of units, such as beads, including without limitation highly porous polymeric beads; glass or silica beads including, but not limited to fused silica (amorphous pure silica), quartz (crystalline pure silica); or other any other suitable beads described herein or otherwise known in the art, which can be packed into a chamber or column, to which the synthesis reagents are delivered. The methods, devices and compositions described herein can be used to scale nucleic acid synthesis methods using microfluidic approaches.

[0094] Microfluidic approaches can be used to for applications of solid phase phosphoramidite chemistry. In some embodiments, mobile solid support units are delivered to one of four chambers in each cycle of an iterative process. In this approach, mobile units to be extended with a particular nucleoside may be delivered and comingled to the same chamber on that particular cycle. After each cycle the units may be redistributed to be delivered again to the appropriate chamber to receive the next base. In some embodiments, units are selected from beads having a diameter and / or size in the range of 10-100 μm. The beads may be monodisperse. Nucleic acids may be synthesized on a plurality of units, including without limitation beads, for example on ten to ten thousand beads or on hundreds of thousands to millions of beads in parallel in a small microfluidic device. Implementation of this approach may comprise one or more of (1) a set-up for encoding hundreds of thousands to millions of units, such as 10-100 μm beads, with of unique barcodes, (2) a set-up for detecting the units while beads are moving at high speeds, (3) a method for directing or distributing beads into the appropriate output chambers on each iteration, and (4) integration of these components in a functional microfluidic system for iterative operation.

[0095] In some embodiments, oligonucleotides are synthesized in the 5′ to 3′ direction. In some embodiments, 5′ to 3′ synthesis is achieved by performing one or more of (i) functionalizing of units; e.g. silanization, amino functionalization, hydroxyl functionalization; (ii) providing photolabile 5′-phosophoramidites, e.g. 3′-NPPOC-deoxyadenosine (N6-benzoyl)-5′-β-cyanoethylphosphoramidite, 3′-NPPOC-deoxycytidine (N4-acetyl)-5′-β-cyanoethylphosphoramidite, 3′-NPPOC-deoxyguanosine (N2-dimethylformamidine)-5′-β-cyanoethylphosphoramidite and / or 3′-NPPOCdeoxythymidine-5′-β-cyanoethylphosphoramidite; (iii) dosing the units with light of a suitable wavelength, e.g. UV light; and (iv) coupling a photolabile 5′ phosphoramidite to the functionalized unit and / or to a nascent oligonucleotide associated with the unit. Oxidation, capping and deprotection steps may be performed similar to 3′ to 5′ phosphoramidite synthesis. (See e.g. Albert et al., Nucleic Acids Research, 2003, Vol. 31, No. 7 e35, DOI: 10.1093 / nar / gng035; Nuwaysir et al., Genome Res. 2002. 12:1749-1755, doi:10.1101 / gr.362402; and Singh-Gasson et al., Nature Biotechnology volume 17, pages 974-78 (1999)).

[0096] In some embodiments, 5′ to 3′ synthesis is achieved by performing one or more of (i) functionalizing of units; (ii) providing phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group (reverse BzNPPOC phosphoramidites); (iii) performing a coupling reaction with the reverse BzNPPOC DNA phosphoramidites, e.g. by irradiating the units with light of appropriate wavelength, e.g. UV light; (iv) performing a capping step; and (v) performing an oxidizing step. Deprotection steps may be performed prior to the addition of new reverse BzNPPOC phosphoramidites. (See e.g. Holz et al., Scientific Reports (2018) 8:15099, DOI:10.1038 / s41598-018-33311-3.)

[0097] Forward and reverse phosphoramidites (i.e., phosphoramidites with a protecting group in the 5′ or 3′ position, respectively) may be purchased from Glen Research, Sterling, Virginia.

[0098] Since microbead barcoding problem had thwarted a number of groups and prevented development of a working device, innovative alternative technology was developed. In various embodiments, methods and compositions described herein comprise a fluidic device in which the beads or other types of units are constrained to narrow fluidic channels, such that they are maintained in a one-dimensional array (FIG. 1-3). This system, in various embodiments, allows the beads or other types units to be identified by their position alone. In some embodiments, beads are loaded into a primary channel. The primary channel may be a capillary or a channel engineered into a suitable substrate. As the beads or other types of units begin the process in a primary channel, they can be pushed, one-by-one, through a distributing mechanism that would direct the beads or other types of units into an appropriate branch channel. Both the primary channel and branch channels may be sized to prevent the beads sliding or squeezing past one another. Once distributed, phosphoramidite chemistry, or other desired chemistry, can take place in the branch channel. After completion of each round of synthesis, the beads or other types of units may be moved in an ordered fashion back into the primary channel for redistributing and a subsequent round of synthesis. In some embodiments, the diameter and / or size of the units and corresponding channels is configured such that units cannot pass each other within a channel or would do so at a rate that is lower than a threshold. For example, units having a diameter and / or size greater than 50% of the width of the channel containing them may be selected.

[0099] A T-junction or flow focusing method may be configured to eject beads or other types of units from the terminus of the primary array and move them towards a router, e.g. a distributor, for example one at a time. Introducing a gap between units may allow for optical detection and routing, e.g. distribution, before the next unit reaches the router. The router may direct the units into one of the branch channels and / or reaction chambers. One or more of available branch channels or reaction chambers may be configured to allow addition of one of the four DNA bases to a nascent nucleic acid, e.g. DNA chain. The distributing mechanism may potentially comprise a multiway router, or a router with two sequential binary routers enabling multiple branchings (FIG. 4). A set of optical detectors could be positioned at the outlets of one or more routers to verify that each unit was distributed to the intended outlet. Once some or all the units have been distributed, a cycle of the phosphoramidite chemistry may be performed in some or all of the branch channels or reaction chambers and an appropriate nucleoside may be added to nucleic acid molecules in or on some or all the units in some or all of the branch channels or reaction chambers. A subsequent cycle can involve a different chemistry, e.g., addition of modified nucleosides or non-phosphoramidite nucleosides, or treatment e.g., a physical or light based treatment. The methods and devices described herein may be used to apply a different reaction or treatment to some or all branch channels or reaction chambers in some or all cycles. In some embodiments, units are redistributed between cycles of reactions or treatments. The cycles of reactions or treatments may be asynchronous for the units held in different branch channels or reaction chambers. For example, if units are held in two or more branch channels, units held in one branch channel may undergo a first cycle of reaction, and subsequently all of the units held in some or all of the branch channels may undergo a second cycle reaction.

[0100] Introduction of the synthesis reagents may be accomplished by using separate reagent ports, e.g. near the beginning of branch channel or reaction chamber outlets. After the completion of a round of synthesis, the units may be recirculated in an ordered fashion back into the primary channel for redistributing and a subsequent round of synthesis. In some embodiments, such recirculation of units comprises reversing the direction of the units backward relative to the direction units entered a branch channel or reaction chamber, thereby causing the units to move into a primary (or main) channel. The process may be repeated as desired, e.g. until the nucleic acid synthesis on all units is complete. In some embodiments, a fluidic device includes an additional output channel to enable synthesis of nucleic acids, e.g. DNA sequences, of different lengths. As modification, e.g. synthesis, on a unit is completed, it may be directed to such an additional output channel and be kept from cycling through the process further. Additional routers, e.g. distributors, and / or sub-channels may be used to handle units that have been incorrectly distributed. Such routers and / or sub-channels may be used to redirect units for redistributing into a correct channel immediately, or directing them into channels where no modifications are made, and then moving these units back into the primary channel before the next cycle so they can be distributed correctly.

[0101] This approach can circumvent the need for a barcoding technology entirely. It can also eliminate the need for a complex and potentially expensive optical detection and image processing system. Instead of a costly system, simple optical detectors may be optionally implemented for counting beads. In various embodiments, beads and other types of units may be processed at high speeds. Further, low cost optical checkpoints may be implemented to verify correct distributing.

[0102] In various embodiments, the order of the mobile units as they are routed within the microfluidic devices described herein is set in a deterministic manner, for example by distributing and releasing the units into and from reaction chambers in a predetermined manner. The position or relative position of specific mobile units may be known or determinable from the path each mobile unit has taken in a prior round of distributing and recombining. In some embodiments, the order of the units is set by tracking the units by detectors operably connected to detect units as they are routed within the microfluidic devices described herein. The devices and methods described herein allow for positional encoding, such that the order of mobile units within the device at a given time and / or location carries information about the path a unit has followed during routing steps. For example, the order of units may be used to determine, which of a plurality of branch channels a unit has been distributed to and / or merged from. In some embodiments, information that was used to determine the order of the beads, such as tracking information, is itself determinative of elements of the routing path that a unit has been routed through. In some embodiments, the devices and methods described herein are configured to route units through a microfluidic device deterministically. The order of units at a given time and / or location within the microfluidic devices described herein, in combination with such a routing algorithm may be used to determine elements of the routing path that a unit has been routed through.

[0103] Elements of a unit's routing path may be determinative of the identity of a compound that was synthesized on a unit as it was routed through the microfluidic devices described herein. More generally, the reaction conditions and / or treatments to which a unit has been subjected to as it was routed through the microfluidic devices described herein, as well as their order, may be determined from the location of the unit. In various embodiments, such location relates to a relative position of a unit within an ordered set of units. Units that have been routed through the microfluidic devices described herein may be mapped to specific routing paths with the use of position information specified relative to other units within the microfluidic device, such as units that are in close vicinity of a particular unit within an ordered set of units.

[0104] In various embodiments, chemical products may be associated with mobile units. The chemical compounds may be in or on the mobile units, they may be tethered or attached, or adsorbed by the mobile unit. The units may be identified by their positional relationship either to each other or to the system. The chemical products associated with each unit may be determined by the history modification procedures applied to each unit. In various embodiments, the absolute or relative position of the units is controlled over time. The positional relationship of the mobile units may be controlled by a variety of suitable methods. For example, the positional relationship may be maintained by ordering the units, for example in a one-dimensional array (1d-array; e.g. single row). This array of units can be split into two or more new branch arrays, which may be one dimensional. The direction of the unit flow through the splits may be controlled. The positional information of the units may be updated with each split. The positional information may include both the new branch array assignment and the position within the new branch array. The various branch arrays comprising the units may be subjected to different modification procedures. A modification procedure may be applied to all of the units in a branch array. The modification procedures and the order of application for modification procedures for each unit may be recorded. After performing modification procedures on the branch arrays, the units in the two or more branch arrays may be merged into a single array. The merging of branch arrays can also be controlled such that the order, branch array history, position, and any procedures applied to units in the new array is recorded. This information may be captured and stored in a computer memory using software specifically built for this purpose. The method may consist of any number of splits, modification procedures, and mergers of branch arrays, wherein the position of and the history of the applied procedures for the units are controlled. The units may be moved through splits, branch arrays, and mergers in series, in parallel, in a loop, or a combination thereof. A large number units, e.g. about, more than, or more than about 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more units, can be directed in a deterministic fashion, having a large number of independent modification procedures applied to produce large targeted or combinatorial libraries of products on the mobile units. Values for the number of units may range between any of the potential values set forth for the number of unit herein. In some embodiments, units are directed through the channels of a microfluidic device without specifically controlling the path for each unit or randomly. Such units may also be tracked and thereby positionally encoded, for example based on the units' relative positions. Tracking information can be used to determine the chemical steps a unit has gone through, for example in split synthesis applications. The products on each unit may be predicted or determined based on the chemical steps the unit has gone through.

[0105] The branch arrays and corresponding modification procedures to be applied to units flowing through may be specifically pre-assigned at every split such that some or all units receive a specific set of modification procedures and are directed appropriately at each splitting event. The series of modifications may be preordained, but assigned to units randomly. In some embodiments, the series of modifications is not preordained. The units may be assigned to a series of modifications deterministically or randomly, e.g. every other unit or an average 50% of units may be directed to a certain path during the splitting event. Regardless of how the assignments are made, the position of units and modification procedures may be recorded.

[0106] Suitable designs for the system and units may be selected in order to enable or enhance features of the methods and compositions relating to the invention. For example, ratios between unit size, height, length, width, diameter, and / or cross-section and / or fluidic channel size, height, width, depth, diameter, and / or cross-section may be selected such that the units would not typically be disarranged or mixed under routine operating conditions, thus maintaining the order of the units within a channel, including without limitation in narrow channels physically restricting mixing or as units are moved within channels in maintained order, for example in in laminar or laminar-like flow. The units can be directed from a single channel, into two or more branch channels by any appropriate mechanism, such as pressure differential, flow focusing (e.g., hydrodynamic focusing), lateral movement of the unit in the laminar flow, valves, gates, routers described in further detail herein, e.g. distributors, or switches of various types (e.g. acoustic, electrophoretic, or photonic) and / or other suitable mechanisms known in the art. Flow focusing may include acoustic focusing and inertial focusing, as described in further detail in U.S. Pat. Nos. 7,340,957 and 9,347,595, both of which are herein incorporated by reference in their entirety. The force inducing the movement of the units through the channels may be from fluidic pressure created by a pump, from electroosmotic forces, or any other transport mechanism known in the art. The input channel or the branch channel, or other channels described in further detail elsewhere herein may be associated with a detector. The detector may be configured to count units, confirm that units were directed into the correct channel, or otherwise track the units and / or the units' relationship to each other or to fiducial marks in the microfluidic device. In some embodiments, units are reordered based on detector read-out, for example when units are erroneously distributed. The detector(s) may be coupled to programs, such as computer programs on a computer configured to accept input from the detector(s). Based on the input from the detector(s), for example when the detector detects certain features, the program may execute certain functions. For example, the detector(s) may be coupled to a feedback loop, such as a feedback loop for controlling the pressure of pumps within or coupled to a microfluidic device. The pressure control may be used to control / adjust the speed of the units. The direction or speed of clumped or adhered units may be adjusted. For example, units may be directed into a particular channel so that they can be separated or isolated from the remainder of the units. Detectors of any suitable type may be used in various embodiments of the invention, including without limitation laser or LED detectors, or CCD based devices. Two or more channels, such as branch channels, may converge into one output path. The movement of the units may be controlled and / or positions of the units in the output channel may be updated as the units are combined in the output path. In one embodiment, units from multiple channels may be merged into a single channel by directing units from one channel through a merging branch point first and subsequently directing the units from a second channel through the merging branch point. The absolute or relative positions of some or all of the units may be tracked or determined accordingly.Channels

[0107] Within a microfluidic system designed to hold ordered sets of, for example channels sized to hold i d-arrays of units, the capacity of the channel may be set based on the average diameter, size, or cross-section of the units. The channels may be narrow to physically constrain the units as they move through the channel such that a unit cannot physically pass the unit ahead or behind it. For example, the channel width may be between 1 to 2 times the average or nominal diameter and / or size of the units. In some embodiments, units are constructed of a rigid non-compliant material, such as glass or rigid polymer, e.g. polystyrene crosslinked with divinyl benzene, or other suitable polymer know in the art. In some embodiments, units constructed from rigid non-compliant material are held or flowed in the microfluidic channels described herein. Units constructed from such rigid non-compliant material may be maintained in order by physically preventing them from passing each other inside channels that are narrow enough to constrict them. Channels may be broad enough to allow for the passage of units constructed from rigid non-compliant materials. In some embodiments, the ratio of average or nominal unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, more than, or more than about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or more. In some embodiments, the ratio of average or nominal unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, less than, or less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55 or less. In some embodiments, the ratio of unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel falls within a range bounded by any of the foregoing values, for example 0.45-0.99, 0.45-0.95, 0.45-0.90, 0.45-0.85, 0.45-0.80, 0.45-0.75, 0.45-0.7, 0.45-0.65, 0.45-0.6, 0.5-0.99, 0.5-0.95, 0.5-0.90, 0.5-0.85, 0.5-0.80, 0.5-0.75, 0.5-0.7, 0.5-0.65, 0.5-0.6, 0.5-0.55, 0.55-0.99, 0.55-0.95, 0.55-0.90, 0.55-0.85, 0.55-0.80, 0.55-0.75, 0.55-0.7, 0.55-0.65, 0.55-0.6, 0.6-0.99, 0.6-0.95, 0.6-0.90, 0.6-0.85, 0.6-0.80, 0.6-0.75, 0.6-0.7, 0.6-0.65, 0.6-0.6, 0.65-0.99, 0.65-0.95, 0.65-0.90, 0.65-0.85, 0.65-0.80, 0.65-0.75, 0.65-0.7, 0.65-0.65, 0.7-0.99, 0.7-0.95, 0.7-0.90, 0.7-0.85, 0.7-0.80, 0.7-0.75, 0.75-0.99, 0.75-0.95, 0.75-0.90, 0.75-0.85, 0.75-0.80, 0.8-0.99, 0.8-0.95, 0.8-0.90, 0.8-0.85, 0.85-0.99, 0.85-0.95, or 0.85-0.90. Values for the channel ratio may range between any of the potential values set forth for the channel ratio herein.

[0108] In some embodiments where units are constructed from a compliant material, such as droplets, slugs, immiscible volumes, hydrogels, or compliant polymers, the ratio of average or nominal uncompressed unit diameter and / or size (as measured outside of the channel) to channel width may be substantially larger than 1. In some embodiments, the ratio of average or nominal uncompressed unit diameter and / or size (e.g. as measured outside of the channel) to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, more than, or more than about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.30, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.70, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.5, 3.0, 3.5, 4.0 or more. In some embodiments, the ratio of average or nominal uncompressed unit diameter and / or size the ratio of average or nominal uncompressed unit diameter and / or size (as measured outside of the channel) to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel is about, less than, or less than about 4.0, 3.5, 3.0, 2.5, 2.0, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.90, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 055 or less. In some embodiments, the ratio of unit diameter and / or size to channel width for all or a portion, such as 90%, 95%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more, of units flowing through the channel falls within a range bounded by any of the foregoing values, for example 0.5-4, 0.5-3.5, 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.95, 0.5-1.85, 0.5-1.8, 0.5-1.75, 0.5-1.7, 0.5-1.65, 0.5-1.6, 0.5-1.55, 0.5-1.5, 0.5-1.45, 0.5-1.4, 0.5-1.35, 0.5-1.3, 0.5-1.25, 0.5-1.2, 0.5-1.15, 0.5-1.1, 0.5-1.05, 0.5-1, 0.5-0.95, 0.5-0.9, 0.5-0.85, 0.5-0.8, 0.5-0.75, 0.5-0.7, 0.5-0.65, 0.5-0.6, 0.5-0.55, 0.55-4, 0.55-3.5, 0.55-3, 0.55-2.5, 0.55-2, 0.55-1.95, 0.55-1.85, 0.55-1.8, 0.55-1.75, 0.55-1.7, 0.55-1.65, 0.55-1.6, 0.55-1.55, 0.55-1.5, 0.55-1.45, 0.55-1.4, 0.55-1.35, 0.55-1.3, 0.55-1.25, 0.55-1.2, 0.55-1.15, 0.55-1.1, 0.55-1.05, 0.55-1, 0.55-0.95, 0.55-0.9, 0.55-0.85, 0.55-0.8, 0.55-0.75, 0.55-0.7, 0.55-0.65, 0.55-0.6, 0.5-0.55, 0.6-4, 0.6-3.5, 0.6-3, 0.6-2.5, 0.6-2, 0.6-1.95, 0.6-1.85, 0.6-1.8, 0.6-1.75, 0.6-1.7, 0.6-1.65, 0.6-1.6, 0.6-1.55, 0.6-1.5, 0.6-1.45, 0.6-1.4, 0.6-1.35, 0.6-1.3, 0.6-1.25, 0.6-1.2, 0.6-1.15, 0.6-1.1, 0.6-1.05, 0.6-1, 0.6-0.95, 0.6-0.9, 0.6-0.85, 0.6-0.8, 0.6-0.75, 0.6-0.7, 0.6-0.65, 0.65-4, 0.65-3.5, 0.65-3, 0.65-2.5, 0.65-2, 0.65-1.95, 0.65-1.85, 0.65-1.8, 0.65-1.75, 0.65-1.7, 0.65-1.65, 0.65-1.6, 0.65-1.55, 0.65-1.5, 0.65-1.45, 0.65-1.4, 0.65-1.35, 0.65-1.3, 0.65-1.25, 0.65-1.2, 0.65-1.15, 0.65-1.1, 0.65-1.05, 0.65-1, 0.65-0.95, 0.65-0.9, 0.65-0.85, 0.65-0.8, 0.65-0.75, 0.65-0.7, 0.7-4, 0.7-3.5, 0.7-3, 0.7-2.5, 0.7-2, 0.7-1.95, 0.7-1.85, 0.7-1.8, 0.7-1.75, 0.7-1.7, 0.7-1.65, 0.7-1.6, 0.7-1.55, 0.7-1.5, 0.7-1.45, 0.7-1.4, 0.7-1.35, 0.7-1.3, 0.7-1.25, 0.7-1.2, 0.7-1.15, 0.7-1.1, 0.7-1.05, 0.7-1, 0.7-0.95, 0.7-0.9, 0.7-0.85, 0.7-0.8, 0.7-0.75, 0.75-4, 0.75-3.5, 0.75-3, 0.75-2.5, 0.75-2, 0.75-1.95, 0.75-1.85, 0.75-1.8, 0.75-1.75, 0.75-1.7, 0.75-1.65, 0.75-1.6, 0.75-1.55, 0.75-1.5, 0.75-1.45, 0.75-1.4, 0.75-1.35, 0.75-1.3, 0.75-1.25, 0.75-1.2, 0.75-1.15, 0.75-1.1, 0.75-1.05, 0.75-1, 0.75-0.95, 0.75-0.9, 0.75-0.85, 0.75-0.8, 0.8-4, 0.8-3.5, 0.8-3, 0.8-2.5, 0.8-2, 0.8-1.95, 0.8-1.85, 0.8-1.8, 0.8-1.75, 0.8-1.7, 0.8-1.65, 0.8-1.6, 0.8-1.55, 0.8-1.5, 0.8-1.45, 0.8-1.4, 0.8-1.35, 0.8-1.3, 0.8-1.25, 0.8-1.2, 0.8-1.15, 0.8-1.1, 0.8-1.05, 0.8-1, 0.8-0.95, 0.8-0.9, 0.8-0.85, 0.85-4, 0.85-3.5, 0.85-3, 0.85-2.5, 0.85-2, 0.85-1.95, 0.85-1.85, 0.85-1.8, 0.85-1.75, 0.85-1.7, 0.85-1.65, 0.85-1.6, 0.85-1.55, 0.85-1.5, 0.85-1.45, 0.85-1.4, 0.85-1.35, 0.85-1.3, 0.85-1.25, 0.85-1.2, 0.85-1.15, 0.85-1.1, 0.85-1.05, 0.85-1, 0.85-0.95, 0.85-0.9, 0.9-4, 0.9-3.5, 0.9-3, 0.9-2.5, 0.9-2, 0.9-1.95, 0.9-1.85, 0.9-1.8, 0.9-1.75, 0.9-1.7, 0.9-1.65, 0.9-1.6, 0.9-1.55, 0.9-1.5, 0.9-1.45, 0.9-1.4, 0.9-1.35, 0.9-1.3, 0.9-1.25, 0.9-1.2, 0.9-1.15, 0.9-1.1, 0.9-1.05, 0.9-1, 0.9-0.95, 0.95-4, 0.95-3.5, 0.95-3, 0.95-2.5, 0.95-2, 0.95-1.95, 0.95-1.85, 0.95-1.8, 0.95-1.75, 0.95-1.7, 0.95-1.65, 0.95-1.6, 0.95-1.55, 0.95-1.5, 0.95-1.45, 0.95-1.4, 0.95-1.35, 0.95-1.3, 0.95-1.25, 0.95-1.2, 0.95-1.15, 0.95-1.1, 0.95-1.05, 0.95-1, 1-4, 1-3.5, 1-3, 1-2.5, 1-2, 1-1.95, 1-1.85, 1-1.8, 1-1.75, 1-1.7, 1-1.65, 1-1.6, 1-1.55, 1-1.5, 1-1.45, 1-1.4, 1-1.35, 1-1.3, 1-1.25, 1-1.2, 1-1.15, 1-1.1, or 1-1.05. Values for the channel width and / or channel ratio may range between any of the potential values set forth for the channel width and / or channel ratio herein.

[0109] The units may be flowed from or to areas where positional ordering is maintained by a physical dimensional constraint, as described in further detail elsewhere herein, into, through, or from portions of the device not having constricting dimensions for physically constraining mixing of units. However, ordered flow of units may be maintained under suitable operating conditions, such as by the application of laminar or laminar-like flow. Operating conditions for maintaining positional order may be maintained at all times, or some of the times, during operation of the device. In some embodiments, microfluidic devices described herein have areas of expansions, gradual or abrupt, in the channel width in some or all directions, for example, a narrow channel with a circular cross section transitioning to a channel with a rectangular cross section and a wide aspect ratio. Such expansions may increase one or more dimension of a channel such that mixing of units flowing therein is not constrained by the physical dimensions of the channel. Such areas of expansions may also include corners and / or chambers of various aspect ratios. Without being bound by theory, in laminar or streamline, flow, parallel layers of fluid flow without disruption between the layers. Positional ordering of units may be maintained as the units are moved through an expansion, by moving the units in ordered flow, such as in laminar or laminar-like flow conditions sufficient to maintain ordering of units. Flow in such expansions need not necessarily be laminar, but maintenance of positional ordering may be established by adjusting flow conditions empirically, in accordance with the various embodiments herein. In various embodiments, devices and methods described herein maintain ordered flow of units, including without limitation while moving units in less than perfect laminar flow or while holding beads, for example as limited by the rate of diffusion. In various embodiments, units are flowed from a first area of the device where position is maintained via physical constraints, as described, into a second area, where order can be maintained by the application of suitable fluidic conditions during the operation of the devices described herein. For example, in such a second area, the channel cross-section width at its widest point may be between 2 to 1000 times the average diameter and / or size of the units. The channel cross-section width at its widest point may be about, more than, or more than about 2, 2.2, 2.4, 2.5, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 500, 1000 or more times the average or nominal diameter and / or size of the units. The channel cross-section width in its widest dimension may fall within a range bounded by any of the foregoing values, including for example 2-2.5, 2-4, 2.5-3, 2-5, 3-3.5, 3.5-4, 3.5-5, 4-4.5, 4.5-5. 5-10, 10-25, 25-50, 50-75, or 75-100, 100-200, 200-500, 500-1000 times the nominal or average diameter and / or size of the units. Units may be moved further into a third area of the device having constricting dimensions allowing for maintaining the order of units physically. In various embodiments, units are held in a designated order in channels that expand and / or constrict. For example, units held in a channel having a sufficiently small width to physically constrict unit mixing may be moved into another region of the channel or another channel having greater width in at least one dimension, such as a width that is about, is more than, or is more than about 2, 2.2, 2.4, 2.5, 2.8, 3, 3.2, 3.4, 3.5, 3.6, 3.8, 4, 4.2, 4.4, 4.5, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 500, 1000 or more times the average or nominal diameter and / or size of the units. Units in such an expanded region of a channel may be kept in a designated order, for example by keeping units in laminar flow. Similarly, units kept in designated order within a region of a channel that is too wide for physically constricting mixing may be moved into another region of the channel or another channel having a width that is narrow enough to physically constrict mixing, for example a channel width that is about, is less than, or is less than about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.05, 1.02, 1.01, or 1 times the average or nominal diameter and / or size of the units therein. Such channel widths may be about, less than, or less than about 0.99, 0.95, 0.9, 0.8, 0.7, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1 times the uncompressed (e.g. as measured outside of the channel) average or nominal diameter and / or size of the units therein or less and may still be able to flow compressible or compliant units through. Such channel width transitions may occur in a transition length that is about, is less than, or is less than about 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, or less. Values for the channel width transitions may range between any of the potential values set forth for the channel width transitions herein.

[0110] In some embodiments, the channel width or mean channel width is or is greater than 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1000 μm or greater. In some embodiments, the channel width or mean channel width is or is less than 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, or less. Channels of the devices described herein may have a channel width or mean width within a range bounded by any of the dimensions listed herein, for example 1-5 μm, 3-8 μm, 5-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 1-100 μm, 100-200 μm, 200-300 μm, 300-400 μm, 400-500 μm, or 100-500 μm, 500-1000 μm. In some embodiments, the height to width aspect ratio of the channel(s) can be 1:100 or greater, e.g. 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:19, 1:18, 1:17, 1:16, 1.15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1.5, 1:4, 1:3, 1:2, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, or greater. The height to width aspect ratio can also be less than 1:1, e.g. less than 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or less. In some embodiments, the height to width aspect ratio of the channel(s) can be 10:1 or less, e.g. 100:1, 90:1, 80:1. 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1 or less. The height to width aspect ratio can also be greater than 1:1, e.g. greater than 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1 80:1, 90:1, or 100:1 or more. The height to width aspect ratio of a channel may fall within a range bounded by any of the values listed above, for example the height width aspect ratio may be between 1:100 and 1:20, 1:20 and 1:1, 1:1.1 and 1.5:1, or 1:3 and 3:1.

[0111] The channel(s) length(s) can be about, greater than, or greater than about 0.01 millimeter (mm), 0.1 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 90 mm, 100 mm, 15 centimeters (cm), 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 90 cm, 100 cm, 1.5 meter (m), 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, or more. The channel lengths may fall in a range bounded by any of the dimensions listed herein, e.g. within 1-10 mm, 10-15 mm, 15-20 mm, 20-25 mm, 30-35 mm, 35-45 mm, 45-50 mm, 50-55 mm, 55-60 mm, 60-65 mm, 65-70 mm, 70-75 mm, 75-80 mm, 80-90 mm, 90-100 mm, 10-15 cm, 15-20 cm, 20-25 cm, 30-35 cm, 35-45 cm, 45-50 cm, 50-55 cm, 55-60 cm, 60-65 cm, 65-70 cm, 70-75 cm, 75-80 cm, 80-90 cm, 90-100 cm, 1-2 m, 2-3 m, 3-4 m, 4-5 m, 5-6 m, 6-7 m, 7-8 m, 8-9 m, 9-10 m. The channel(s) length(s) can be about, less than, or is less than about 10 m, 9 m, 8 m, 7 m, 6 m, 5 m, 4 m, 3 m, 2 m, 100 cm, 90 cm, 80 cm, 70 cm, 60 cm, 50 cm, 40 cm, 30 cm, 20 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.01 mm, or less. Values for the channel length may range between any of the potential values set forth for the channel length herein.

[0112] In some embodiments, the length of one or more channels is selected based on the number of units in the device or the number of units that are designated to fit in the channel. Unit sizes are described in more detail elsewhere herein including without limitation in the Unit section in paragraph 129. The channel length may be selected to fit a number of units in a range bounded by any of the values listed herein, e.g., about 1-1E7 units, 1-10, 10-50, 50-100, 50-1E5, 100-500, 100-5E5, 100-1E7, 500-1E4, 1E4-5E4, 5E4-1E5, 1E5-5E5, 5E5-1E6, 1E6-5E6, or 5E6-1E7 units. The channel length may be selected to fit about, more than, or more than about 1, 10, 50, 100, 500, 1E4, 5E4, 1E5, 5E5, 1E6, 5E6, 1E7, or more units. A channel length may be selected to fit about, less than or less than about 1E7, 5E6, 1E6, 5E5, 1E5, 5E4, 1E4, 500, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 units. A branch channel length may be selected to fit a number of units in a range bounded by any of the values listed herein, e.g., 1-1E7 units, 1-10, 10-50, 50-100, 50-1E5, 100-500, 100-5E5, 100-5E7, 500-1E4, 1E4-5E4, 5E4-1E5, 1E5-5E5, 5E5-1E6, 1E6-5E6, or 5E6-1E7 unit lengths. A branch channel length may be selected to fit about, less than, or less than about 1E7, 5E6, 1E6, 5E5, 1E5, 5E4, 1E4, 500, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 units. A branch channel length may be selected to fit about, more than, or more than about 1, 5, 10, 20, 30, 40 50, 100, 500, 1E4, 5E4, 1E5, 5E5, 1E6, 5E6, 1E7, or more units. Values for the branch channel length may range between any of the potential values set forth for the branch channel length herein.

[0113] The units may be spaced from each other with spacer lengths about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the nominal or average size and / or diameter of the units or more. The channel(s) may be selected to have sufficient length to accommodate a desired number of units, for example 1-1E7 units with a spacer length of about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the length of a unit between each unit or more. The channel(s) may be selected to have sufficient length to accommodate 1-1E7 units with a spacer length of about, less than, or less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times the length of a unit between each unit or less. The channel(s) may be selected to have sufficient length to accommodate 1-1E7 units with spacer lengths falling within a range bounded by any of the spacer length values described herein, for example 1-1000, 1-100, 2-25, 3-40, 4-10, 5-100, 6-30, 7-100, 8-100, 9-10, 10-15, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 spacer length between each unit. Values for the spacer length may range between any of the potential values set forth for the spacer length herein.

[0114] The channel cross-section shape may be square, rectangle, oval, circular, half-circular, or any other suitable shape. Microfluidic channels can be linear, serpentine, or have another suitable shape or length to enable channels with large unit capacities. Unit capacities of 1E6, 1E7 or higher may be achieved using suitable channel configurations on relatively small fluidic chips.

[0115] According to the various embodiments, channels can be used as reaction chambers where modification procedures are used to modify the products, or in some cases the units. Modification procedures may comprise any chemical, physical, optical, or mechanical method. Various embodiments of the invention ensure that modification procedures do not interfere with the arrangement of the units. Chemical reagents may be flowed as liquids or gasses through the fluidic channel(s) containing the units. The characteristics or diameter and / or size of the channel or the units may be selected to enhance the flow of chemical reagents, or the effectiveness or efficiency of chemical procedures. For example, the channels may be constructed from glass, chemically resistant polymers, or non-resistant polymers or coated with the same. In various embodiments, the channels are chemically resistant to the modification procedures applied. Units may be constructed from any suitable material, such as controlled pore glass, plastic, or any suitable polymer. In various embodiments, the size distribution of units may be selected to leave space for fluids to flow over the units while in the channel. In various embodiments, there may be no space for fluid to flow over the units. Treatments and chemical reactions described in further detail elsewhere herein may be performed without requiring space for fluid to flow over the units within the channels of the microfluidic devices described herein. For example, treatments comprising the application of heat or light may be performed without such spaces.

[0116] The present invention may include reaction chambers. Various regions within the microfluidic devices described herein, for example branch channels, may be utilized as reaction chambers. Reaction chambers may be enclosed by valves located in or at the end of a channel. Reaction chambers may also be valve-less and the pressure or flow of carrier fluid and / or reagents controlled by pumps with inlets or outlets connecting to the reaction chamber. The units can be flowed from one reaction chamber to another directly or through one or more channel(s). The size of the reaction chamber can vary and may depend on the spacing or size of the valves or pump inlets / outlets defining the reaction chamber(s) and the dimensions, e.g. width, height, diameter, or cross-section of the reaction chamber(s). The size of the reaction chambers can be about, at least, or at least about 10 pl, 20 pl, 30 pl, 40 pl, 50 pl, 60 pl, 70 pl, 80 pl, 90 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 100-200 pl, 200-300 pl, 300-400 pl, 400-500 pl, 500-600 pl, 600-700 pl, 700-800 pl, 800-900 pl, 900-1000 pl, 1 nl, 2 nl, 3 nl, 4 nl, 5 nl, 6 nl, 7 nl, 8 nl, 9 nl, 10 pl nl, 20 nl, 30 nl, 40 nl, 50 nl, 60 nl, 70 nl, 80 nl, 90 nl, 100 nl, 200 nl, 300 nl, 400 nl, 500 nl, 600 nl, 700 nl, 800 nl, 900 nl, 1 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, or more. The size of the reaction chambers can be less than or less than about 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 1000 nl, 900 nl, 800 nl, 700 nl, 600 nl, 500 nl, 400 nl, 300 nl, 200 nl, 100 nl, 90 nl, 80 nl, 70 nl, 60 nl, 50 nl, 40 nl, 30 nl, 20 nl, 10 nl, 9 nl, 8 nl, 7 nl, 6 nl, 5 nl, 4 nl, 3 nl, 2 nl, 1 nl, 900 pl, 800 pl, 700 pl, 600 pl, 500 pl, 400 pl, 300 pl, 200 pl, 100 pl, 90 pl, 80 pl, 70 pl, 60 pl, 50 pl, 40 pl, 30 pl, 20 pl, 10 pl, or less. Those of skill in the art will appreciate that the reaction chambers may have a size that falls within any range bound by any of these values, for example 10-50 nl, 10-100 nl, 50-100 nl, 100-200 nl, 200-300 nl, 300-400 nl, 400-500 nl, 500-600 nl, 600-700 nl, 700-800 nl, 800-900 nl, 900-1000 nl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 1-10 μl, 10-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. Values for the reaction chamber may range between any of the potential values set forth for the reaction chamber herein.

[0117] Channels in which modification procedures occur may have one or more inlet or outlet ports and / or valves. Reagents may be delivered through valve or port into and out of the channel. These inlet or outlet ports and valves may be configured or suitably occluded so as to prevent units from becoming trapped or disarranged. The units may be held in a channel, for example during a modification procedure, by one or more closed, occlusive, or porous valves, gates, switches, or by magnetic fields. Units having permanent or inducible magnetic properties may be employed to utilize their interaction with magnetic fields. A modification procedure may be operated on some or all of the units in a particular channel. In some cases, the selected modification procedure does not cause a change in the unit or the product associated with the unit. Zero or more modification procedures may be applied to units in given channel. Different channels of a fluidic device may be configured to enable distinct modification procedures that can be applied, either sequentially or simultaneously, to the units in the respective channels. Channels may split more than once before converging, separate modification procedures can be applied to any channel.

[0118] In various embodiments, all units intended to receive the application of the same reaction condition(s) are kept in a single channel designated for the application of such reaction condition(s). In some embodiments, units designated to receive the application of the same reaction condition(s) are distributed into a plurality of channels or reaction chambers, including for example branch channels.

[0119] The microfluidic device may contain branch points where the channel splits or divides into multiple channels or outlets. The branch points may comprise about, at least, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more channels or outlets, including without limitation branch channels or reaction chambers. Values for the branch points may range between any of the potential values set forth for the branch points herein. One or more branch points may be arranged sequentially. The branch channels or outlets may have 2-dimensional or 3-dimensional arrangements. For example, a branch point may split a first channel into two or more branch channels in the X, Y plane, resulting in a 2-dimensional planar channel arrangement within the device. Or, a branch point may split a first channel into two or more branch channels in and / or out of the X, Y plane. In such an arrangement, one or more branch channels in a first set may be in one plane A with the portion of the first channel immediately adjacent to the branch point, while the branch-point adjacent portions of one or more branch channels in a second set may be in a different plane than plane A, for example, perpendicular to place A, resulting in a 3-dimensional branch-point channel arrangement within the devices described herein. In some embodiments, one or more channels in the devices described herein are non-linear, for example such devices may have the shape of a spiral, or other curve.Routing of Units

[0120] The microfluidics device described herein can be configured to route units through the device. Routing of units may comprise holding units, moving units, distributing units into channel(s) or branch channel(s) and / or merging units from two or more channels or branch channels to one or more channel(s). The device can also be configured to merge units from two or more channel(s) or branch channel(s) to one or more channel(s). In various embodiments, routing comprises distribution. Units within microfluidic devices described herein may be routed from p locations, e.g. channels, to p+i locations within the microfluidic device, where p, i>0, through a distributor. These p+i locations may be channels generally referred to as branch channels herein. In various embodiments, routing comprises merging. Units within microfluidic devices described herein may be routed from q locations, e.g. channels, into q−j locations, where q, j, q−j>0, through a merger. These q-j locations may be channels generally referred to as merger channels herein. In some embodiments, p is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, p is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, p is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, i is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, i is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, i is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, q is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, q is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, q is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. In some embodiments, j is, is at least, or is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 120, 30, 40, 50, 60, 70, 80, 90, 100, or more. In some embodiments, j is, is at most, or is at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less. In some embodiments, j is between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 85-90, 90-95, or 95-100. Values for p, q, j, and / or i, may fall within a range bounded by any of the potential values set forth the p, q, j, and / or i, herein. Routing may comprise the movement of units within a channel, or from one location in a fluidic device to another, or from a first channel to a second channel, where the axis of flow of the first channel may be the same as the second, or alternatively the axis of flow of the first may be at any angle, for example, 45° or 90°, to that of the axis of flow of the second. Distribution may comprise the movement of units from a first channel into a branch channel via a branch point, from one or more branch channel(s) or reaction chamber(s) into one or more other channel(s). Merging may comprise the reverse of distribution. Units may be merged by moving units from q locations within a microfluidic device, e.g. q branch channel(s) or reaction chamber(s) via one or more branch point(s) and into q-j locations within the microfluidic device where q>j, for example into a first channel from which the units had been distributed.

[0121] The microfluidic device described herein can be configured to steer / route units via any appropriate mechanism known in the art, including but not limited to, mechanisms for generating and modulating fluid flow (e.g., as in electroosmotic mechanisms described below), generating or modulating fluidic pressure, moving mechanical mechanisms, static or non-moving mechanical features, or non-moving force generating mechanism. Routers constructed according to such routing mechanisms or any other suitable mechanism known in the art, may be configured and used to move or route units within a first channel, move or route units from a first channel to a second channel, distribute units from a first channel into two or more branch channels, and / or merge units from two or more branch channels into a first or second channel. The microfluidic device described herein may have one, two, or multiple routing mechanisms.

[0122] Fluidic pressure modulation routing mechanisms may include, but are not limited to, mechanisms that increase or decrease the fluidic pressure at one or more locations within a fluidic device. Fluidic pressure modulation mechanisms may comprise any appropriate mechanical device known in the art such as fluidic pumps, gas pressure driven pumps, manual syringes, electronically controlled syringe pumps, electroosmotic pumps, diaphragm pumps, gear pumps, peristaltic pumps, electrohydrodynamic pumps, or any combination thereof. The devices described herein may contain one or more fluidic pressure modulating mechanisms of the same type, or of different types. Fluidic pressure modulating mechanisms may or may not be under specific electronic control, and may have feedback control to ensure appropriate pressure delivery. The fluidic pressure modulating mechanisms may operate independently or be under synchronized control. Not wishing to be bound by theory, units may be moved, flowed, advanced, reversed, held, stopped, directed, and / or redirected in the device by applying increased or decreased relative or absolute pressure to fluids and / or units in the device.

[0123] In one embodiment, routing by the microfluidic device is implemented with inclusion of elements for providing pressure-driven crossflows, for example where a pressure-driven crossflow is configured using fluidic pressures, for example externally generated pressures, which may be gated by one or more valves. The valve(s) associated with the pressure-driven crossflow can be integrated with the microfluidic device (e.g., on-chip), or distinct from the microfluidic device (e.g., or off-chip). In an example, the valve(s) include pneumatic values, for example pneumatic valves actuated at 4 bar at over 300 Hz; however, other pressure levels and frequencies of actuation can be implemented by the microfluidic devices described in further detail herein. Furthermore, the medium used to drive the crossflow can be liquid or gas (e.g. air). Routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000 Hz or more.

[0124] Moving mechanical routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids within a fluidic device. Methods and devices described herein may utilize any suitable moving mechanical routers known in the art, including but not limited to, plugs, pistons, gates, flippers, valves, pins, ratchets, or any combination thereof. Units may be held by a closed mechanical router of a device and / or released upon opening of the mechanical router. Moving mechanical routers may be configured to apply a force either directly to the units, and / or to the fluid in a device described herein such that units may be moved, stopped, held, directed, and / or redirected in the device.

[0125] In one embodiment, routing by the microfluidic device is implemented with inclusion of a mechanical actuator includes a cross-channel fluidically connected to two or more chambers, wherein each of the two or more chambers has at least one compliant side (e.g. the roof) which may be deflected to change the volume of the chamber. In a two-chamber embodiment, a first force applied to the compliant side of the chamber displaces fluid towards the flow channel, and a second force applied to the compliant side of the second chamber displaces fluid from the flow channel into the second chamber. Such a configuration can be connected to two sides of a routing junction of a microfluidic device such one chamber can operate in a “pulling mode” and the other chamber can operate in “pushing mode”, whereby flow can be configured to be purely across the junction, rather than adding fluid flow along the primary flow direction in a manner that opposes or adds to the primary flow. In variations, the embodiment of the mechanical actuator can include elements for providing forces including one or more of: magnetic forces (e.g., with permanent or electromagnets coupled to the compliant side of the chamber); piezeoelectric-provided forces (e.g., with extending and contracting piezoelectric actuators coupled to the compliant side of a chamber, “buckling”-type actuators); mechanical actuators (e.g., mechanical solenoids coupled to and configured to push and pull the compliant side of a chamber); external pressure sources (e.g., gas pressurizers configured to apply and release pressure by one or more pneumatic valves); and other actuators. In some embodiments, the mechanical actuator includes elements for providing forces using surface acoustic waves, transient surface acoustic waves, electrophoresis, dielectrophoresis, micromechanical valves, optical tweezers, and / or thermal vapor bubbles, for example vapor bubbles created by laser absorption or by electrical heating. Routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000 Hz or more.

[0126] In another embodiment, a mechanical component includes a deflector (e.g., flipper valve) that steers flow, beads, or other units toward a desired path or channel. In examples, the deflector(s) can be actuated by one or more of: microelectromechanical systems (MEMS) actuators integrated into a channel network, magnetic actuators providing magnetic fields that actuate magnetic flippers, mechanical actuators / servos, and other actuators for manipulating flippers integrated with the channel network.

[0127] Static or non-moving mechanical routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids within a fluidic device. Such routers may utilize any appropriate static mechanical mechanism known in the art, including but not limited to pillars, grooves, wedges, walls, scallops, holes, cups, divots, sieves, selective stops (e.g. allow fluids to pass, but units are held back), dams, weirs or other similar mechanism, or any combinations thereof. The microfluidic device described herein may comprise one or multiple static or non-moving mechanical routers. The microfluidic device may comprise a single type of static router, for example one or more selective stop, or two or more types of static routers, for example one or more dam and one or more pillar. Such examples are not meant to be limiting. Static or non-moving mechanical routers may be configured to apply a force either directly to the units, or to the fluid in the device such that units may be moved, stopped, held, directed, and / or redirected in the devices described herein.

[0128] Non-moving force generating routers include, but are not limited to, routers that can be configured to move, control, or alter the movement of units or fluids, including without limitation compositions, such as oligomers within such fluids, within a fluidic device (e.g., thermal vapor bubbles). Such routers may use any appropriate static mechanical mechanism known in the art, including but not limited to electrophoresis, dielectrophoretic, acoustophoresis, electroosmosis, magnetophoresis, gravity, or any combination thereof (see e.g., Wyatt Shields C. et al, Lab Chip 2015 15(5):1230-1249, incorporated herein by reference in its entirety). In one embodiment, the units are routed via magnetophoresis. Non-moving force generating routers may be configured to apply a force directly to the units, and / or apply a force to or through the fluid in the device such that units may be moved, stopped, held, directed, and / or redirected in the devices described herein.

[0129] In one embodiment, a non-moving force generating router includes one or more magnets (e.g., permanent magnets, electromagnets) configured to route units responsive to magnetic fields or magnetic field gradients (e.g., paramagnetic, superparamagnetic beads may be moved by a magnetic field gradient). Magnetic fields and / or field gradients can be generated by devices integrated within or outside the microfluidic devices described herein. Permanent magnets may be actuated toward and / or away from desired regions of the system to affect fields. Electromagnets may be transitioned between on and off states to affect fields. In embodiments, pointed magnets and / or electromagnet cores are used to produce high-field gradients.

[0130] In a related embodiment, microfluidic devices described herein may be configured to implement ferrofluid actuation. A ferrofluid (e.g., a suspension of fine magnetic particles in a liquid medium which is immiscible with the liquid containing beads or other units) is actuated (e.g., by a permanent magnet, by an electromagnet) in response to applied magnetic fields. Such an embodiment can include an operation mode that moves the ferrofluid toward an applied magnetic field in order to displace fluid in a channel, thereby forming a ferrofluid “piston”. Such an embodiment can additionally or alternatively include an operation mode where the ferrofluid is configured to become rigid when subjected to a magnetic field. Upon becoming rigid, the ferrofluid may be used to block one or more of the exits of a routing junction, thereby causing the flow and / or units, e.g. beads, to be routed towards an un-blocked channel.

[0131] In some embodiments, routers described herein comprise or are implemented in cooperation with one or more electroosmotic pumps. Electroosmotic pumps according to the various embodiments described herein, may be controlled electrically in order to generate a flow across a steering junction. For example, an electroosmotic pump can be coupled to or otherwise interface with multiple sides of a steering junction such that it “pulls” on a first side and “pushes” on another side (e.g., in a closed loop). In such a configuration, flow can be configured to be purely or primarily across the junction, as opposed to requiring added fluid flow along a primary flow direction (which would oppose or add to the primary flow). Furthermore, such a configuration can provide fast response speeds. (e.g., providing responses in less than 1 ms).

[0132] In some embodiments, routers described herein comprise or are implemented in cooperation with electrophoretic steering apparatus configured to provide an electric field in solution. For example, units, e.g. coupled to nucleic acid (e.g., DNAs, DNA fragments) can be inherently negatively charged in aqueous solution and manipulated by electrophoretic forces that route such units in a desired manner.

[0133] In some embodiments, routers described herein comprise or are implemented in cooperation with dielectrophoretic steering apparatus configured to provide an electric field in solution. For example, a dielectrophoretic apparatus can be configured to manipulate particles that electrically polarize under the influence of an electric field (e.g., high frequency field on the order of kHz). Without being bounded by theory, if the units' polarization is significantly different than that of the surrounding medium, a net dielectric force is exerted on the particles, which can be used for steering the unit (e.g., at a rate of 400 Hz). Dielectric forces may be applied to units in solution directly; additionally or alternatively. In some embodiments, dielectric forces are applied to beads encapsulated in aqueous droplets in an oil. Dielectrophoretic routing of units may be performed at rates of, of about, or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400 Hz or more. In some embodiments, dielectrophoretic routing of units is performed at rates less than 500, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 Hz or less. Dielectrophoretic routers may be configured to route units within the microfluidic devices described herein at a rate that falls within a range bounded by any of the foregoing values, e.g. 100-400 Hz, 10-300 Hz, 20-500 Hz, etc.

[0134] In another embodiment, routers described herein comprise or are implemented in cooperation with charged droplet steering apparatus that encapsulates beads in aqueous droplets in an oil stream. The droplets may be given a net charge. The routers may be used to route the charged droplets, for example steer them at a sorting junction, such as by affecting attraction toward and / or repulsion by charged plates. In various embodiments, routers acting on charged droplets may be configured to route units within microfluidic devices described herein at, at about, or at greater than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 Hz or higher. Routers acting on charged droplets may be configured to route units within the microfluidic devices described herein at rates less than 2000, 1000, 900. 800. 700. 600, 500, 400, 300, 200, 100, 75, 50, 25, 20, 10 Hz or less. Routers acting on charged droplets may be configured to route units within the microfluidic devices described herein at a rate that falls within a range bounded by any of the foregoing values, e.g. 100-1000 Hz, 10-2000 Hz, 20-500 Hz, etc.

[0135] In another embodiment, routers described herein comprise or are implemented in cooperation with an electrostrictive apparatus. For example, electrostrictive apparatus may comprise sources of positive and negative potentials applied to opposing sides of a layer of an elastomeric polymer. Without being bound by theory, such positive and negative potentials may be used to apply forces that cause the polymer to compress. The elastomeric polymer may be coupled to or integrated with a wall of a chamber, such that the applied forces cause the chamber volume to expand and / or contract. In various embodiments, multiple chambers configured to be under the control of electrostrictive apparatus may be coupled to a flow channel via a cross channel (e.g., as described above). A first chamber that may be actuated to pull fluid from the flow channel into the first chamber. If a second chamber fluidically connected to the cross-channel junction is deactivated, fluid may be released from the second chamber to the flow channel.

[0136] In another embodiment, routers described herein comprise or are implemented in cooperation with thermal bubble forming apparatus. Thermal generation of microbubbles can rapidly generate gasses that displace liquid volume. Microbubbles may be used for routing of units, (e.g. beads) in a flow stream, for example, by displacing liquid around a unit and thereby creating a flow stream. In related embodiments, routers comprise or are implemented in cooperation with electrochemical bubble forming apparatus. Electrochemical generation of microbubbles can rapidly generate gasses that displace liquid volume and can be used for routing of units (e.g. beads) in a flow stream.

[0137] Various embodiments of routers described herein comprise microheaters or microactuators configured to generate bubbles. Use of microheaters and microactuators for bubble generation is further described in aus der Wiesche, S. Rembe, C., Maier, C., and Hofer, E. P. Dynamics in Microfluidic Systems with Microheaters (1999) and Chen, C., Wang, J. and Solgaard, O. Micromachined bubble-jet cell sorter with multiple operation modes. Sensors and Actuators B. 2006; 117: 523-529, both of which are herein incorporated by reference in their entirety. See also, Vercruysse, D., Liu, C., Dusa, A. d Wijs, K., Majeed, B. Miyazaki, T. Peeters, S., and Lagae, L. A High-Speed Miniaturized Cell Sorter with Lens-Free Imaging and Thermal Bubble Based Jet Flow Sorting. 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2014: 382-384; UK Patent Application No. GB 2561587; and European Patent Application No. EP2602608, all of which are herein incorporated by reference in their entirety. Without being bound by theory, bubbles generated within a fluid of substantially laminar flow may cause a displacement of the fluid and / or unit(s) within the fluid laterally with respect to the direction of flow, e.g. by a force that disturbs the fluid and / or unit(s) within the fluid laterally with respect to the direction of flow.

[0138] In some embodiments, routers described herein comprise a vortex element or elements configured to generate a vortex in a flow stream within a desired distance from the microactuator, e.g., within about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm or more from the microactuator. Without being bound by theory, a vortex generated downstream from a microactuator configured to generate bubbles in the fluid moving along a channel may be used to amplify the displacement of the fluid and / or unit(s) within the fluid and / or the force that disturbs the fluid and / or unit(s) within the fluid. For example, a displacement and / or force generated by a microactuator configured to generate bubbles upstream from the vortex element may be amplified by the vortex. In some cases, the vortex elements configured to generate a vortex may comprise a recess, turn, and / or protrusion in a channel within a microfluidic device (e.g., a triangular recess on the channel wall). The generated vortex may travel downstream with the unit to be sorted and may cause routing of the unit into a branch channel. In some embodiments the vortex generating element is located between a microheater or microactuator configured to generate bubbles, and a branch channel.

[0139] In some embodiments, microfluidic devices described herein comprise a microactuator configured to generate bubbles, one or more vortex elements configured to generate a vortex and / or a branch point configured to route a unit in flow into one of a plurality of branch channels, located within 100 μm to 10 mm along the direction of flow. The path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may have a length of at least 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 1000 μm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, or more. The path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may have a length of at most 10.0 mm, 9.0 mm, 8.0 mm, 7.0 mm, 6.0 mm, 5.0 mm, 4.5 mm, 4.0 mm, 3.5 mm, 3.0 mm, 2.9 mm, 2.8 mm, 2.7 mm, 2.6 mm, 2.5 mm, 2.4 mm, 2.3 mm, 2.2 mm, 2.1 mm, 2.0 mm, 1.9 mm, 1.8 mm, 1.7 mm, 1.6 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.0 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, or less. The length of the path comprising a microactuator, one or more vortex elements configured to generate a vortex, and / or a branch point configured to route a unit into a branch channel may fall within a range bound by any of the foregoing values, e.g. 1.6-8.0 mm, 750 μm-2.3 mm, or 100 μm-3.5 mm.

[0140] In some embodiments, microfluidic devices comprising routers comprising or implemented in conjunction with a thermal bubble forming apparatus comprise one unit delivery channel and one or more (e.g. two) fluid delivery channels that all open into a switch chamber with two branch outlet channels. The fluid delivery channels may be connected to a common fluid source. In some embodiments, the fluid delivery channels terminate in a firing chamber and a nozzle connected to the switch chamber. In some embodiments, one or more microactuator(s) (e.g. microheater(s)) is incorporated into the wall of the firing chamber. The microactuator may be used to generate a bubble, e.g. following application of an electrical pulse. Growth of the bubble may force the incoming fluid from a fluid delivery channel to pass through the nozzle and form a jet that pushes a selected unit toward the desired (e.g. the farther) branch outlet channel. Timing of the application of the electrical pulse may be calibrated to cause incoming units to enter into a designated branch outlet channel.

[0141] The routers comprising or implemented in cooperation with a thermal bubble forming apparatus described herein may be fabricated using any suitable method known in the art, including without limitation photolithography and deep reactive ion etching (DRIE) to form microfluidic channels and etch on an opposite side of a silicon wafer housing the microfluidic channels openings providing connections to the channels and access to actuator pads. Optical access may be provided by any suitable material known in the art, such as by Pyrex wafers covering the microfluidic channels. Bubble actuator circuits may be patterned on a suitable medium known in the art, e.g, Pyrex. In one example, trenches about 1 μm deep are etched on Pyrex wafers using hydrofluoric acid (HF); a layer of about 400 Å titanium is sputtered on the Pyrex and patterned by a lift-off process to construct microheaters; conducting wires are made by successive sputtering and lift-off of 3000 Å gold; an insulating layer of 3000 Å silicon nitride is deposited on Pyrex by plasma-enhanced chemical vapor deposition (PECVD) and patterned by plasma etch; and Pyrex wafers are anodically bonded to Si wafers. Heaters, conductors and / or insulators may be patterned in the Pyrex trenches. The construction of routers comprising or implemented in cooperation with a thermal bubble forming apparatus (e.g., bubble-jet distributors) are described in Chen, C., Wang, J. and Solgaard, O. Micromachined bubble-jet cell sorter with multiple operation modes. Sensors and Actuators B. 2006; 117: 523-529, which is incorporated herein by reference in its entirety.

[0142] In another embodiment, routers described herein comprise or are implemented in cooperation with acoustic steering apparatus. An acoustic steering apparatus may be configured to generate sound / pressure waves and apply such waves to flow within the system (e.g. in a way that produces standing waves of low and / or high pressure). Standing acoustic waves may be used to route units, e.g. beads in the microfluidic devices described herein. In some embodiments, transient surface acoustic waves are used to route units.

[0143] In another embodiment, routers described herein comprise or are implemented in cooperation with electrowetting apparatus that generates electric fields / electric charges. Electric fields and / or charges may be configured to modulate surface tension at corresponding liquid-gas, liquid-solid, and / or liquid-liquid interfaces. Modulation of surface tension may be used to maintain or release pressure gradients that cause fluids to remain still or move under resulting modified pressures provided by the electrowetting apparatus, thereby routing units (e.g. beads in fluid communication with the modulated fluids).

[0144] Routers as described further herein may be configured to merge one or more units from different channels or branch channels. For example, a router may be configured to merge one or more units from a first channel to a second channel, from two or more channels into a single channel, or from two or more branch channels to a second channel. A single type of router or any combination of routers may be used in a single device. The sequencing of moving specific individual units or sets of units into to specific locations within a device, or from one channel to another, or from two or more branch channels into a single destination channel may be controlled by a single type of router or a combination of different types of routers as described in further detail herein. The movement of units into one or more channels may be verified by one or more detectors.

[0145] The microfluidic device described herein may be configured to distribute one or more units from one channel to one or more channels or branch channels via any appropriate distribution mechanism known in the art. The devices described herein may comprise one or more types of distributors. Distributors in a microfluidic device can be configured to stop, hold, direct, or redirect units or fluid in the device. A distributor may be used to close off sections of the channel(s) or branch channels, or to impede progress of the units through or into a channel or branch channel.

[0146] Distributors in a microfluidic device may be configured to distribute one or more units from a primary channel into one or more branch channel(s) based on the positional order of the unit(s) in the primary channel. Distributors may also be configured to distribute one or more units into one or more branch channel(s) based on a label on a unit. The distribution of one or more units to a branch channel may be predesignated according to an intended sequence of reactions and / or treatments. The intended sequence of reactions and / or treatments may be preassigned to the one or more unit. The channels to which the one or more units may be distributed may also be randomly assigned to the one or more unit. Methods to distribute one or more units into a channel or branch channel include, but are not limited to, altering the position of a unit within the laminar or laminar-like flow at or before a branch point; the presence of one or more moving or non-moving mechanical devices at or before a branch point to direct units into a channel or branch channel; any method that alters the amount or pressure of the fluid flow through branch channels such that units are directed into one or more branch channel(s), or any combination thereof or any other suitable method known in the art. The correct distribution of one or more units into one or more branch channel(s) may be verified using detectors. Incorrectly distributed units may be subjected to an error correction mechanism described elsewhere herein, for example, by directing and / or holding one or more units into a side channel and / or redirecting the one or more units in a side channel back into a primary channel holding positionally ordered units, and / or any other suitable error correction mechanism known in the art.

[0147] In some embodiments, the units are distributed by altering the position of the unit within the fluid. Such methods can alter the position of the unit within ordered flow, for example within laminar or laminar-like flow, of a channel. Lateral movement of a unit within flow may cause the unit to be directed into a desired channel at a branch point, typically the channel located at the same side as the unit's relative position within flow prior to the branch point. Methods that alter the position of a unit within the flow include the application of electrostatic or electrokinetic forces such as electrophoresis, dielectrophoresis, and electroosmotic flow; acoustic forces such as bulk standing waves, standing surface acoustics waves, and traveling waves; optical manipulation(s) or optical radiation with focused laser beam(s), also known as optical tweezers; application of side flow or cross flow at an angle to the flow direction of a unit to move the unit laterally within the flow; gravity; magnetophoresis if the units contain ferromagnetic materials; flow focusing; via the application of any other suitable type of force known in the art; or combinations thereof. In some embodiments, application of side flow or cross flow is performed by application of pressure, electroosmosis, or displacement via pistons or actuators, such as those comprising piezoelectric, electrostatic or electroactive polymers, or pumps such as electoosmotic pumps.

[0148] In some embodiments, the units are distributed by moving mechanical distributors. Moving mechanical distributors that may be configured to distribute units include, but are not limited to, rotary valves, ratchet mechanisms, pins, flippers, gates, flow switching mechanisms, or channel actuation via application of heat to a thermoreversible gelation polymer.

[0149] In some embodiments, the units are distributed by methods that alter the fluidic pressure of a channel, including without limitation a branch channel. This method can be used to cause increased or decreased fluid from one channel to flow into another specified channel at the branch point. For example, as the relative pressure is increased in one channel and decreased in a second, connecting channel, the carrier fluid and units carried therein can be directed into the second channel with the lower relative pressure.

[0150] Routers, e.g. distributors, having suitable configurations as described in further detail herein may also be used as mergers to merge units from at least q channels into q-j channel(s), where q>j. For example, units from two channels may be merged into one merger channel, or units from four channels may be merged into three, two, or one merger channels. Differential pressures may be utilized to cause release of units from two or more branch channels into one or more channels in a designated order. By application of a lower relative pressure into a first branch channel, units therein may be kept from entering the branch point and / or an adjacent merger channel while units from a second branch channel leading to the same branch point may be released from the second branch channel into and / or past the branch point. Such units may be routed into the merger channel prior to the release of units from the first branch channel into the branch point and / or merger channel.

[0151] In various embodiments, dedicated routers, e.g. distributors, are used to facilitate the movement and / or merging of mobile units. For example, a router, e.g. a distributor, placed at the branch point of two channels can be configured to direct one or more unit into one or more channel(s) or branch channel(s) during distribution. In the reverse direction, the same router may block, hold, or impede the movement of units from a first branch channel while allowing the movement of units from a second branch channel into a single channel, thus allowing the controlled and / or orderly distribution of units as well as the controlled and / or orderly merging of units. Distribution of units into branch channels may comprise distributors that act on one or more units with spacing between them. Units may be merged from p channels into p-b channels, where p>b, via use of any router, e.g distributor, to route one or more units in a first channel and then route one or more units from a second channel.

[0152] In various embodiment, microfluidic devices and systems comprise one or more of the following: high-speed routers, e.g. distributor(s), for directing units into one of the multiple branch channels, e.g. for parallel synthesis; high-speed unit counting sensor(s) configured to detect units prior to a distribution step; and device integration that combines discrete components, for example unit router(s), unit detector(s), multiple capillaries, and / or reagent mixing chips into a complete device.

[0153] The position of the units in the device may be maintained by a variety of methods. For example, the position of the units in the device may be maintained by placing physical constraints on the units in a channel(s) to preserve the relative position of the units or by spacing the units in a channel(s) under continuous flow or by a combination of both within the same device. To place physical constraints on units, a channel width may be selected to be sufficiently narrow so that units cannot pass one another in the channel. To maintain order of units in flow, e.g. in laminar or laminar-like flow, the units may be separated in continuous or stopped flow with sufficient space between the units that they do not pass one another during the flow or during stopped flow. While uncontrolled migration of the units due to factors like, but not limited to, diffusion, or sedimentation may eventually cause units to pass one another, stopped flow for short periods of time can maintain order of sufficiently spaced units for desired periods of time.

[0154] The microfluidic device described herein may also correct unit position errors introduced during the operation of a microfluidic device described herein, for example during operation for nucleic acid synthesis. Additional routers and channels may be added to the system to handle units that have been incorrectly distributed. Units incorrectly distributed at a first router may be routed into a second channel where correct distribution can be performed immediately. For example, a channel comprising a loop can return a unit to a position before the distribution router such that the unit can be correctly routed. Units can also be routed into branch channels and held for either the remainder of device operation, or they can be held temporarily and subsequently routed back to into position to be distributed.

[0155] In some cases, two or more neighboring units may exchange position while not affecting other units on either side of the exchanged units. In various embodiments, such units getting out of positional order are identified by a detector. This type of error may result in incorrect reactions, treatments, or modifications being applied to the units, e.g. incorrectly synthesized molecules on affected units. In some embodiments, this error occurs at less than 0.000001 times, 0.00001 times, 0.0001 times, 0.001 times, 0.0025 times, 0.005 times, 0.0075 times, 0.01 times, 0.025 times, 0.05 times, 0.075 times, 0.1 times, 0.25 times, 0.5 times, 0.75 times, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 30 times per unit per 100 cycles of modification.

[0156] In some cases, one or more units may be incorrectly distributed through mis-routing at a branch point. In various embodiments, mis-routed units can be identified by a detector. In some embodiments mis-routing can be detected in the channel in which reactions or treatments occur. In some embodiments, mis-routing can be detected after mis-routing by detectors placed after the branch point. In some embodiments detection of the mis-routing event can occur at any point between the branch point and the channel in which the reactions or treatments occur. The effect of this type of an error may be limited to only the mis-routed units. Subsequent units may be correctly routed, and only the mis-routed unit may be affected by the mis-routing. In some embodiments, the mis-routing is detected, and the positions of all units is updated accordingly so that the history of each unit is known and units with the desired sequence of treatments can be identified and / or from those without the desired sequence of treatments, e.g. nucleic acid synthesis steps.

[0157] In various embodiments, additional routers and channels may be added to the microfluidic device system to hold units that have been incorrectly distributed. In some embodiments, a mis-routed unit may be detected and routed into a branch channel for holding until the unit can be routed back, for example for further distribution. In some embodiments, treatments and chemical reactions are reserved from mis-routed units held in such channels. Units can also be routed into branch channels and held for either the remainder of device operation or discarded. Units incorrectly distributed at a first router, e.g. a distributor, may be re-routed into a second channel where correct distribution can be attempted immediately, such as a channel comprising a loop that returns a unit to a position before the distributor such that another attempt at correctly routing the unit can be made. In various embodiments, the positional information of the mis-routed units and all other units is updated, such that the position and history of all or a subset of the units throughout the device remains known. In some embodiments, these types of errors may be tracked or corrected such that they do not result in a loss of correct sequence of treatments or modifications applied to or to be applied to some or all units.

[0158] In some embodiments, this type of a mis-routing error occurs at, less than or less than about 0.000001 times, 0.00001 times, 0.0001 times, 0.001 times, 0.0025 times, 0.005 times, 0.0075 times, 0.01 times, 0.025 times, 0.05 times, 0.075 times, 0.1 times, 0.25 times, 0.5 times, 0.75 times, 1 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 30 time per unit per 100 cycles of modification. Values for the error rates may range between any of the potential values set forth for the error rates herein. In some embodiments, mis-routed units may escape detection. This type of error may result in incorrect synthesis history for units that are out of positional order. In some embodiments, labeled units capable of labeling units, for example beads that can be colored with pigment or imbued with fluorescent properties, are used to verify routing. Detectors at any point in the device or on any cycle of operation may be used to verify such labeled units are in the expected relative position. In one embodiment for example, one in 100 beads in a device may be labeled with a fluorescent dye. During device operation, the relative positional ordering of these labeled and distinguishable beads may be verified against their expected position based on predesignated routing paths for each of the units. In some embodiments, the verification occurs in reaction channels after each cycle of device operation. In other embodiments verification occurs on each cycle in the initial channel prior to distribution. In further embodiments verification occurs only once after all cycles are complete and all modifications have taken place.

[0159] In various embodiments, devices and systems described herein are operated for multiple cycles, where all or substantially all of the units within a microfluidic device are returned to a common area, such as a channel. Unit detection, identification of mis-routing events, corrective routing may be performed one or more times during each cycle of operation.Unit Spacing

[0160] In various embodiments, units are held and moved together in a group having units adjacent to each other in a channel. This “stacked regime” may comprise units that are held or flowed in direct contact with (e.g. end-to-end and / or with their geometric centers offset) or in close vicinity of each other. In various embodiments, the order of units within a channel is maintained by the restrictive width of the channel holding the units, preventing units from swapping positions outside of their order. The ratio of unit diameter and / or sizes to channel diameter, cross-section, or widths can be selected to maintain positional ordering and / or to prevent wedging of units within a channel which may lead to clogging.

[0161] Without wishing to be bound by theory, units moving through a microfluidic device in a stacked regime can contact each other and the channel at acute angles, creating a force that may push the units into the channel walls. This may result in the likelihood of the units wedging and clogging the channel. Such forces may become so great as to distort or compress the units such that the units stop moving in the channel. In addition, imperfection in the unit surface may likewise prevent movement through the channel. Without wishing to be bound by theory, a solution to units clogging in the stacked regime includes the use of straight and sufficiently smooth channels, and / or units that are sufficiently smooth and / or round. Channels that are straight and sufficiently smooth can support movement of beads in the stacked regime. In addition, unit spacers may be incorporated into the microfluidic devices described herein to separate stacked beads in channels with changing dimensions, e.g. at width transitions, or at branch points.

[0162] Units in the stacked regime may be abutting or touching one another in the channel(s). In some embodiments, units are less than 1 unit length apart in the direction of the flow, e.g. due to the offset geometric centers within a channel. Units may be a fraction of a unit length apart. In some embodiments, units are about, less than, or less than about 2, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05 or fewer unit lengths apart, center to center, in the direction of the flow. Center to center unit spacing in the direction of the flow may fall within any range bound by the foregoing values, including for example 0.1-0.2, 0.1-1, 0.2-0.3, 0.2-1.5, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, or 0.9-2 unit lengths. Values for the unit spacing may range between any of the potential values set forth for the unit spacing herein.

[0163] In various embodiments, units are separated by space from each other. This “separated regime” may facilitate proper distribution by allowing various routers, e.g. distributors, to act on units individually, without interference from other units; may allow units to navigate various features or aspects of the device that could briefly or temporarily slow or impede the movement of a unit such as a corner, constriction, edge, expansion, or combination thereof without risk of clogging due to interference or contact by adjacent units; and may allow units to move to and from areas of the device in ordered flow, e.g. in laminar or laminar-like flow. Flow-based unit ordering, e.g. in laminar or laminar-like flow can be used to allow the use of channels that are greater in width than those allowed in a stacked regime. Ordered flow may be maintained in separated regime in channels having greater widths than widths that allow for maintaining unit order by physically constraining unit mixing, including without limitations widths that are about, more than, or more than about 2 times the width of unit size.

[0164] In a flow-based unit ordering regime, units may be maintained within channels having widths that are about, more than, or more than about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times the average or nominal diameter and / or size of the units. Values for the channel widths may range between any of the potential values set forth for the channel widths herein.

[0165] Also provided herein are methods for separating units. A spacer or ejector device may be configured to apply a fluidic shear force that results in a first unit accelerating away from a second unit as the first unit passes through the spacer or ejector device. The acceleration of the first unit may introduce space and / or additional fluid volume between the first and second unit.

[0166] Units may be moved through a channel feeding into a unit spacer in various configurations, including, without limitation, individually or as a stacked column. When a unit reaches a unit spacer, e.g. at a T-intersection, an in-line spacer channel, or any suitable cross-channel geometry, units may be separated by the additional flow, or “cross flow,” in the main channel. The additional flow(s) for spacing fluids (e.g., for a T-intersection, for a cross-channel, for an inline-spacer channel for other geometries, etc.) entering from side channels may be created without additional pressure sources. For instance, a bypass channel with low fluidic resistance may be used to redirect fluid upstream of a stacked unit (e.g. bead) column and into the spacer. The lower fluidic resistance may be configured to yield a higher flow rate through the bypass channel than along the unit stack. Increased flow rates may be used to induce high shear and a degree of unit separation. Unit separation may be influenced by channel geometries near the spacer.

[0167] Additionally or alternatively, in associated embodiments, the systems described herein comprise one or more spacers (e.g., spacers having independent pressure control a bypass channel) fluidly interfaced with the channels of a microfluidic device. Such spacers may be used to restack beads or other units in a controlled manner. In operation, such a spacer may be used to reduce the spatial extent of a collection of beads or other units in a portion of the system. In various embodiments, such spacers are configured in conjunction with or without unit (e.g. bead) stops. In more detail, the system can allow fluid flow, with beads or other units, through such spacers in reverse, such that one or more beads can be returned in reverse manner through the spacer to allow restacking or re-ordering of beads in a controlled manner. Additionally or alternatively, such spacers may be used with a bypass channel and an element (e.g., valve, check-valve, variable flow restriction unit, one-way flow restriction unit, etc.) that prevents return flow through the bypass channel, such that beads or other units can be removed from a stack, and not re-stacked. Any of the above spacer embodiments can include wall geometries designed to ensure that beads enter the exit channel without jamming. In one example of a wall geometry that mitigates jamming, the wall geometry can include a narrow gap or slit in the channel wall (e.g., primary channel wall, cross-flow channel wall, etc.). Such a gap or slit may have a width that is less than the diameter of one unit, e.g. bead or other characteristic dimension of a bead or other unit. In examples, the gap or slit width can be less than 20 nm, 100 nm, 500 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, etc. Those of skill in the art will appreciate that the slit or gap width may have a value that is within any range bound by any of these values, for example 20-100 nm, 100-500 nm, 500-1000 nm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, etc., with associated tolerances as described above.

[0168] In one example, units entering a unit spacer having a cross-channel geometry from a feeding channel may enter into a cross-flow incoming laterally to the unit's flow. FIG. 22D provides an illustrative implementation of a unit spacer with cross-flow geometry constructed in accordance with the embodiments herein. The cross-flow may be generated by flow from opposing or substantially opposing directions. The cross-flow may be perpendicular or substantially perpendicular or have a component of velocity perpendicular or substantially perpendicular to the units' path through the unit spacer. The cross-flow may be provided by two or more channels leading into the cross-channel geometry of a unit spacer. A first unit may flow past the cross-channel geometry, followed by a mix of fluid from each side of the cross-flow. In some embodiments, the pressures in channels generating the cross-flow are adjusted such that they are equal and greater than the pressure in the downstream portion of the entering unit's path and less than the pressure in the feeding channel. The pressures in channels generating the cross-flow need not be equal. Unequal flows may be used according to various embodiments, for example to bias flowing units laterally with respect to the units' direction of flow. Suitable pressures, pressure differentials, and / or flow rates, flow rate differentials for causing a desired movement of a unit within the microfluidic devices described herein may be selected as described in further detail elsewhere herein or as is otherwise known in the art.

[0169] A spacing may be generated between the first unit and a second unit entering the unit spacer subsequent to the first unit by the mix of fluid from each side of the cross-flow being introduced between the first and the second channel as they move past the unit spacer. In some embodiments, e.g. for a T-intersection type unit spacer, the cross-flow is provided by one channel. The spacing introduced between units may be used to facilitate subsequent distribution of each unit at a branch channel, various embodiments of which are described in further detail elsewhere herein, by allowing that the router, e.g. a distributor, act on units individually for each distribution event. Therefore, entry of a plurality of units into a router at once may be prevented by introducing a space between units moving in channels of the devices described herein.

[0170] Units may also be spaced from each other in the channel. The units may be spaced by a spacer length of about, more than, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 50,000, 100,000 or more unit diameter and / or sizes apart. The units may be spaced by a spacer length of about, less than, or less than about 100,000, 50,000, 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less unit diameter and / or size apart. The spacer length between units may fall within any range bounded by the foregoing limits, including without limitation, between 1-10, 20, 20-30, 30-50, 50-100, 100-250, 250-500, 500-1000, 1000-2500, 2500-5000, 5000-7500, 7500-10,000, 10,000-50,000, 50,000-100,000 unit diameter and / or sizes. The units may be spaced by a spacer length of about, more than, or more than about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 14 μm, 15 μm, 16 μm, 18 μm, 20 μm, 25 μm, 50 μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, 10,000 μm, 50,000 μm, 100,000 μm or more. The units may be spaced by a spacer length of about, less than, or less than about 100,000 μm, 50,000 μm, 10,000 μm, 9000 μm, 8000 μm, 7000 μm, 6000 μm, 5000 μm, 4000 μm, 3000 μm, 2000 μm, 1000 μm, 900 μm, 800 μm, 700 μm 600 μm, 500 m, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm or less. The spacer length between units may fall within any range bounded by the foregoing limits, including without limitation between 0-10 μm, 20 μm, 20-30 μm, 30-50 μm, 50-100 μm, 100-250 μm, 250-500 μm, 500-1000 μm, 1000-2500 μm, 2500-5000 μm, 5000-7500 μm, 7500-10,000 μm, 10,000-50,000 μm, 50,000-100,000 μm. Values for the unit spacing may range between any of the potential values set forth for the unit spacing herein.Pressure Differentials

[0171] In various embodiments, units flowing through the channels and branch points of the microfluidic devices described herein, may be routed in a designated direction by adjusting pressures and / or flow rates within channels that connect through branch points. FIG. 31 shows exemplary pressure settings in channels connected through one branch point. Without being bound by theory, fluid within microfluidic devices flows down a gradient in pressure. Further, in various channel configurations, pressure drops continuously along the direction of flow. Further without being bound by theory, flow rates through channels of microfluidic devices correlate with the pressure differential between two points (Ptop−Pbottom) divided by the channel length between such two points (FIG. 31A).

[0172] In a branch point of a main channel intersecting with a branch channel (FIG. 31B-F), pressures at locations a distance away from the branch point may be adjusted to set a pressure value at the branch point P0. In FIG. 31B, pressures are adjusted such that Ptop>Pbottom in the main channel and the pressure value at the branch point P0 is equal to Pbranch (P0=Pbranch). In FIG. 31C, pressures at the corresponding locations are adjusted such that Ptop>Pbottom and P0>Pbranch, leading to flow from top to bottom of the main channel as well as from branch point into the branch channel. In FIG. 31D, pressures at the corresponding locations are adjusted such that Ptop>P0>Pbranch and Pbottom>P0>Pbranch, leading to flow from the top and the bottom of the main channel into the branch channel from the branch point. In FIG. 31E, pressures at the corresponding locations are adjusted such that Ptop>Pbranch>P0>Pbottom, leading to flow from the top of the main channel as well as the branch channel toward the bottom of the channel. In FIG. 31F, pressures at the corresponding locations are adjusted such that Pbranch>P0>Ptop and Pbranch>P0>Pbottom, leading to flow from the branch channel to the top as well as the bottom of the main channel. Pressure differentials can be created by setting pressures in a variety of location within microfluidic devices described herein to route flow and / or units carried therein in designated directions following a pressure gradient.

[0173] FIG. 32 provides further exemplary embodiments using pressure differentials to route units within microfluidic devices described herein. A branch point configuration with a main channel and two branch channels B1 and B2 is illustrated describing pressure values at the top and the bottom of the main channel Ptop, Pbottom, respectively, at the intersection of the main channel with the first branch channel B1 and B2, P1, P2, respectively, and at the distal ends of branch channels B1 and B2, PB1, PB2, respectively. FIG. 32B-E provide exemplary values for each of these pressures and resulting flow patterns. For example, flow between the intersection of the main channel with branch channel B1 and the intersection of the main channel with branch channel B2 is governed by the pressure differential P1−P2. Where P1−P2=0, there is no flow between these points (FIG. 32B). Similarly, flow into and out of the first branch channel B1 is governed by the pressure differential P1−PB1; flow into and out of the second branch channel B2 is governed by the pressure differential P2−PB2; and flow between the top of the main channel and the intersection of the main channel with branch channel B1 is governed by the pressure differential Ptop−P1; and flow between the bottom of the main channel and the intersection of the main channel with branch channel B2 is governed by the pressure differential Pbottom−P2.

[0174] Using the pressure differentials exemplified with the pressure values shown in FIG. 32B-E, units may be selectively loaded into branch channel B1 (FIG. 32B) or into branch channel B2 (FIG. 32C). Similarly units can be unloaded selectively from either branch channel. FIG. 32D shows a pressure differential setting for unloading selectively from branch channel B2 toward the top of the main channel. Similar pressure values are set in FIG. 32E as FIG. 32D, except that Pbottom>P2 in FIG. 32E, allowing for flow from the bottom of the main channel past the intersection of the main channel with branch channel B2. Thus, as units are unloaded from branch channel B2 toward the top of the main channel, fluid flowing from the bottom of the main channel is introduced between units creating spacer lengths of fluid (FIG. 32E). In contrast, Pbottom=P2 in FIG. 32D, resulting to no flow from the bottom of the main channel toward the intersection of the main channel with the second channel B2. Such a setting allows the spacing between units to be maintained as the units enter the main channel from the branch channel (FIG. 32D).

[0175] Those skilled in the art will note that similar applications of pressure differentials between various points in microfluidic channels can be used to route, including without limitation to hold, units within microfluidic devices described herein and / or adjust spacing between units.Multi-Branch Channels

[0176] In various embodiments, the microfluidic devices described in further detail herein, comprise routers and branch points with multiple branch channels. In some embodiments, such branch points comprise multiple “sub-branch points” allowing for units to be sorted into branch channels that branch off in consecutive sub-branch points. FIG. 39 depicts an exemplary illustration of a router configured to distribute beads into a plurality of branch channels and / or merge beads from a plurality of branch channels. Incoming units, e.g beads 3901 as depicted in FIG. 39, as they move from the unit spacer 3902 toward the first router 3911, may be routed into a branch channel 3904, via one of the routers 3911, 3912, 3913, 3914. The units may be stopped with a unit stop (e.g. a bead stop) 3921, 3922, 3923, 3924, within and / or at the end of the branch channel. The units may be merged back into the main flow, for example through a spacer 3903, and / or a router 3911, 3912, 3913, 3914. As the units are distributed from and / or merged into the main flow in the main capillary 3910, fluids may be flowed past the branch point, e.g. into a waste reservoir.Units

[0177] The units may be solid or porous. They may or may not carry an attached library product. The units may be glass, polymeric beads, droplets, or cells. The units may be directly modified by the modification procedures described herein. In some embodiments, both a unit and an associated product is modified by one or more modification procedures described herein. Large collections of units can be generated with specific properties such as color, surface chemistries, labels using the various modification procedures described herein. Some or all of the units within a microfluidic device or a channel thereof may be uniquely encoded, without redundancy. The units may be randomly assigned or assigned based on some physical, chemical, or optical characteristic of each unit. A series of modification procedures may be applied sequentially, in a loop or in series, such that each unit is exposed to a particular set of modification procedures. The positional encoding according to the various embodiments of the invention allows the elimination of redundancy. Accordingly, large numbers of physically encoded library units may be generated at low cost. Such library units may be encoded uniquely. Physically encoded library units may be used in downstream procedures. A first procedure where units are physically encoded may be coupled with a second procedure where products are generated on the units, while preserving the positional encoding between the first and second procedures. This approach can be used to associate physical unit encodings with products. By associating the physical encodings with products, the units can be directed into unrelated procedures where the positional information / encoding may be lost, but physical encoding can be detected.

[0178] The units used in various embodiments can be made from a range of materials. In some embodiments, the units are solid. In some embodiments, the units are porous. In some embodiments, the units do not carry an attached library product. The units may be glass, polymeric beads, droplets, bubbles, slugs, or cells. Materials used for beads can include polymers such as polystyrene, melamine resin, polyacrylonitrile, or agarose; hydrogels such as alginate or chitosan; silica, glass, or controlled porous glass (CPG); and metals such as gold, silver, GaAs, GaP, or iron. Silica may be fused silica (amorphous pure silica), quartz (crystalline pure silica), or other generic glass (silica crystalline or amorphous). Many beads can be purchased from vendors such as ThermoFisher or Sigma Aldrich with or without pre-functionalized coatings, including functionalized coatings with reactive chemistries, affinity tags such as biotin or streptavidin, and / or dyes, such as fluorescent dyes. Units may already have a molecule, for example a nucleic acid on their surface while a second, distinct, chemical or molecular compound is added to their surface, or to such molecule during device operation.

[0179] In some embodiments, the units comprise superparamagnetic beads. Without being bound by theory, use of superparamagnetic beads may reduce the number of fluidic connections. In some embodiments, superparamagnetic beads have a high magnetic susceptibility, and are mechanically sound, chemically functionalized, and / or monodisperse.Barcodes

[0180] Units may be barcoded with physical properties, molecular properties, color or pigment, metal, or spectral properties, or any combination thereof. Physical properties include, but are not limited to, etching or shape, or metal bars or deposits. Molecular properties include, but are not limited to, chemical functionalization and chemical compounds, nucleic acids, or biotin or streptavidin affinity tags. Color or pigments include, but are not limited to, fluorescent or non-fluorescent dyes. Barcodes could be used before, after, or during to establish the identity of units prior to commencing operation, during operation to verify the identity of units, or after completion of operation to enable tracking of units after removal from the device and disordering. The identify of barcoded beads may detected and mapped to a unit position so that barcodes need only be read once while positional information is used during operation. Barcodes may be detected at the end of operation to verify correct position.

[0181] Units with or without barcodes or labels may be randomly arranged initially. Units may also be arranged in a known pattern, either due to a deliberate arrangement initially, or as a result of a previous round of synthesis performed using positional encoding. In various embodiments, barcodes (labels) comprise nucleic acids. Barcodes may differ in a variety of chemical or physical feature, such as optical or spectral features, including features described herein or any suitable features known in the art. In some embodiments, barcode differ in more than one type of a feature. For example, a barcode having an optical feature may also comprise an oligonucleotide feature. Multiple features of a barcode can be used in combination to identify one or more features about a unit and / or molecules associated with a unit. For example, one feature of a barcode may be used for identification of a unit. Another feature of a barcode may be used for identification of a target associated with the unit.

[0182] In various embodiments, barcodes are known nucleic acid sequences that allow some feature of a polynucleotide with which the barcode is associated to be identified. In some embodiments, a barcode comprises a nucleic acid sequence that when associated with a target polynucleotide serves as an identifier of the sample from which the target polynucleotide was derived.

[0183] Nucleic acid barcodes can be designed at suitable lengths to allow sufficient degree of identification, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or more nucleotides in length. Multiple barcodes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more barcodes, may be used on the same molecule, optionally separated by non-barcode sequences. In some embodiments, barcodes are shorter than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. In some embodiments, barcodes associated with some polynucleotides are of different length than barcodes associated with other polynucleotides. In general, barcodes are of sufficient length and comprise sequences that are sufficiently different to allow the identification of samples based on barcodes with which they are associated. In some embodiments, each barcode within a plurality of barcodes differ from every other barcode in the plurality at at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more positions.

[0184] In some embodiments, units described herein, such as all or some of the units within a microfluidic device described herein, are associated with one or more of a) a unit-specific barcode uniquely identifying the unit, b) a target specific barcode, and c) a target, e.g. an oligonucleotide. Unit-specific barcodes may be associated with the unit alone or with the unit and the target-specific barcode and / or target. For example, a unit-specific barcode may be attached to a unit and a target-specific barcode and the target-specific barcode may be attached to the target. In some embodiments, unit-specific barcodes are associated with the unit, but are not attached to the target-specific barcode and / or the target. In some embodiments, unit-specific barcodes and target-specific barcodes are linked, e.g. covalently. Target-specific barcodes may be associated with targets in a variety of ways, for example covalently. In some embodiments, a first end of a unit-specific barcode is attached to a unit, the second end of the unit-specific barcode is attached to the first end of a target-specific barcode and the second end of the target-specific barcode is attached to a target. In some embodiments, unit-specific barcodes are attached directly to targets associated with the unit. Target-specific barcodes may also be attached to the target, e.g. through the opposite end of the target from the unit-specific barcode. In some embodiments, unit-associated target-specific barcodes continue to be associated with and / or identify targets after being separated from a unit. In various embodiments, target-specific barcodes for each of the targets on a unit are different. In various embodiments, the unit-specific barcode for each of the targets associated with a unit is the same. Unit-specific barcodes and target-specific barcodes can facilitate methods of target analysis, such as analysis comprising counting. Such analysis may comprise sequencing, hybridization and / or any suitable method known in the art. Such analysis may follow an amplification step. For example, upon amplification of the target along with one or more barcodes described herein, the number of different target-specific barcodes can indicate the number of pre-amplification target molecules. In various embodiments, upon amplification of the target along with one or more barcodes described herein, the number of different unit-specific barcodes is used to analyze the number of units the pre-amplification targets were associated with. Such amplification and / or analysis steps, such as sequencing, may follow a hybrid capture step. For example, nucleic acids in a sample may be hybridized to targets associated with units in a hybrid capture step. One or more barcode sequences may be incorporated to hybridized nucleic acids, for example by a nucleic acid extension elongation reaction. The hybridized nucleic acids may be amplified and / or analyzed. The number of hybridized nucleic acids from the sample may be analyzed by counting the number of different barcodes. The identity of barcodes described herein may be probed using hybridization, sequencing, or any suitable method known in the art. Barcodes described herein may be generated through an oligomer synthesis method (such as nucleic acid synthesis or peptide synthesis), e.g. a number of rounds of monomer, (such as nucleotide) incorporation, such as random incorporation. In some embodiments, barcodes are generated through oligomer synthesis of, of about, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more rounds of monomer incorporation. Barcodes may be synthesized through oligomer synthesis wherein the number of rounds of monomer incorporation falls within any range bound by the foregoing values, e.g. 2-30 rounds, 8-10 rounds etc. In some embodiments, presynthesized barcodes attached to units. Barcodes described herein may comprise oligonucleotides. Synthesis of barcodes may be achieved through oligomer synthesis methods described herein. For example, units may be routed iteratively through reaction chambers or channels of the microfluidic devices described herein. One or more monomers (or dimers or longer building blocks) may be incorporated to the a nascent chain of a barcode in each routing cycle.

[0185] Cells may be bacterial cells or eukaryotic cells, for example cells derived from cell culture, animals, or human subjects, such as cells derived from patient samples. Droplets may be formed by the mixture of immiscible fluids, such as water and oil or other organic solvents, to form an emulsion. Droplet formation for use in microfluidic devices is described in U.S. Pat. Nos. 8,528,589, 9,364,803, 8,658,430, WO2014001781, and US20080286751, which are herein incorporated by reference in their entirety with respect to droplet formation in microfluidic devices.

[0186] The methods described herein can take advantage of beads or other types of units maintaining their order throughout an iterative modification process. In some embodiments, the beads or other types of units cannot pass each other or stick together. The bead or other type of unit distribution may be adjusted to be fairly monodisperse throughout the process. In some embodiments, units are passed through a size selection mechanism generating a population of units that substantially or entirely fall within a predesignated size range, for example by passing units through a size sorter. Units may be size sorted such that the likelihood of detected or undetected undesired unit mixing within the channels of the devices described herein, e.g. within channels having widths that physically prevent mixing of units of a selected average or nominal diameter and / or size, is minimized.

[0187] The beads or other types of units may swell when exposed to non-aqueous reagents, such as toluene, used in DNA synthesis. A swollen bead may stick to capillary walls and impede flow. Various materials, such as divinylbenzene (DVB) cross-linking of polymeric beads can mitigate swelling at an appropriate concentration. Introduction of surfactants may be used to reduce bead / unit adhesion. In more detail, one or more embodiments of the method(s) and / or system(s) described can implement beads or other types of units configured to be non-swelling, non-brittle, monodisperse, and / or porous, and devoid of outlier (in relation to morphological uniformity) beads or other types of units. Outlier units (e.g. beads may be removed by filtering, sieving, suspension processes, processes involving removal of a supernatant of smaller-than-desired particles, or by any suitable method known in the art. FIG. 36 depicts swelling response of divinylbenzene-cross-linked polystyrene (PS) beads in organic solvent versus percent divinylbenzene (DVB) cross-linking agent (in terms of molar ratio between PS and DVB). Beads were put in toluene to quantify swelling response of cross-linked beads in organic solvents. In various embodiments, swelling response of beads can be similarly measured in alternative solvents, such as acetonitrile, toluene, dichloromethane, tetrahydrofuran (THF), pyridine, N-methyl pyrrolidinone (NMP), 2,6-lutidine, carbon disulfide, 1,2-dichloroethane, 1,1-dichloroethane, chloroform, dimethylformamide, dimethylacetamide, dimethylsulfoxide (DMSO), ethylene carbonate, 1,4-dioxane, DME (1,2-dimethoxyethane), nitromethane, methyl tert-butyl ether, methyl ethyl ketone (butanone), or dichloromethane. Without being bound by theory, beads cross-linked in high cross-linker ratios may be brittle and / or be subject to surface solvation (e.g., as in the range from 60-80% DVB cross-linking in FIG. 36), while beads cross-linked in lower cross-linker ratios exhibit acceptable performance in terms of minimal swelling, brittleness, and solvation (e.g., as in the range from 30-60% DVB cross-linker in FIG. 36). Cross-linked beads may be functionalized by a variety of suitable moieties described elsewhere herein or otherwise known in the art, such as by amine or carboxyl groups.

[0188] In various embodiments, units may be filtered by size to provide a uniformly sized unit population. For example, units having a diameter greater than an upper threshold may be filtered out by a suitable method described herein or is known in the art. Amine functionalized polystyrene beads were cross-linked with 35% DVB. Cross-linked beads had a diameter of 34.4 μm with a coefficient of variation of 2.6%. Particles larger than 40 μm were removed by sieving through a 40 μm electroformed mesh. In some embodiments, particles smaller than a desired size are removed by a suitable method described herein or otherwise known in the art, such as by an electroformed mesh of appropriate size. Such size filtering methods may be applied on cross-linked beads of any suitable kind, which may be functionalized by one or more suitable moieties described elsewhere herein or otherwise known in the art, such as by amine or carboxyl groups. In a related variation, small particles, particle fragments, dust, and other contaminants may be removed by suspending the monodisperse PS beads in solution and letting the desired particles settle for a period of time, after which the supernatant with undesired particles and fragments may be removed. In another variation, small particles, particle fragments, dust, and other contaminants may be removed by centrifugation and removal of the supernatant with undesired particles and fragments. In any of the examples and variations described, one or more successive washing steps, optionally intercalated by other filtering steps, such as settling steps, may be implemented to remove undesired particles.

[0189] In embodiments, cross-linking can be performed using one or more of chemical cross-linking agents (e.g., DVB, glutaraldehyde, formaldehyde, epoxy compounds, dialdehyde, dichloroethane, etc.), radiation-induced cross-linking procedures, oxidative cross-linking procedures, and other cross-linking procedures.

[0190] In embodiments, beads used according to the methods described herein are crosslinked at a molar cross-linker ratio of, of about, or of greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher. Beads used according to the methods described herein may be crosslinked at a molar cross-linker ratio of less than 90%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or lower. Beads used according to the methods described herein may be crosslinked at a molar cross-linker ratio that falls within a range bounded by any of the foregoing values (e.g. 10-60%, 3-60%, 45-55%, etc.). Ranges of cross-linking and / or different types of cross-linking agents can be selected to tune swelling behavior, brittleness, and / or solvation as desired. For example, aforementioned parameters may be selected to limit swelling to, to about, or to less than 10%, 9% 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less.

[0191] Units, such as bead units can range in size according to the various embodiments described herein. For example, all or substantially all (e.g. more than 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.99%, 99.995%, 99.999% or more) units used in the methods and devices described herein may have a diameter and / or size from about, at least, or at least about 20 nm, 100 nm, 500 nm, 1000 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 35 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm. Those of skill in the art will appreciate that the unit diameter and / or size may have a value that falls within any range bound by any of these values, for example 20-100 nm, 100-500 nm, 500-1000 nm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm. The coefficient of variation for the size or cross-section of the units can be about, at least, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more. The coefficient of variation for the size or cross-section of the units can be about less than, or about less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. The units can also be oval. Droplet volume may be about, at least or at least about 10 femtoliters (fl), 100 fl, 1 pl, 10 pl, 100 pl, 500 pl, 1 nanoliter (nl), 10 nl, 50 nl, 100 nl, 300 nl, 400 nl, 500 nl, 600 nl, 700 nl, 800 nl, 900 nl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 50, pl, 100 μl, or more. The droplet volume may fall in a range bounded by any of the foregoing values, e.g. 10-100 femtoliters (fl), 100-1000 fl, 1-10 picoliters (pl), 10-100 pl, 100-500 pl, 500-1000 pl, 1-10 nanoliter (nl), 10-100 nl, 100-200 nl, 200-300 nl, 300-400 nl, 400-500 nl, 500-600 nl, 600-700 nl, 700-800 nl, 800-900 nl, 900-1000 nl, 1-10 μl, 10-50 μl, or 50-100 μl. Values for the unit or droplet size may range between any of the potential values set forth for the unit or droplet size herein.

[0192] In various embodiments, screening procedures may be applied to a library of products (or a subset thereof having selected properties) associated with units for which positional encoding is maintained. The positional encoding of the units can be used to identify products of interest. For example, after a library of products is made, the associated units, arranged in a 1d-array with known absolute or relative positions, can be exposed to a set of screening reagents. In various embodiments, screening reagents are delivered in the same or a similar manner as the reagents for modification procedures. Screening reagents may be moved through channels holding the products to be screened, such as channels holding the associated units in an ordered 1d-array. The units or the associated products may be evaluated for their reactivity to screening interactions, for example by optical analysis of the units in place or by flowing the units past a detector, such as an optical or magnetic detector. Units or associated products displaying features of interest, such as an ability to interact with a target compound, can be detected. A product associated with a unit detected for a screened feature can be identified, for example by the position of the unit.

[0193] In some embodiments, the physical encoding on the units may be associated with the units' positional encoding within a system. For example, the physical encoding of units may be read once in the beginning or end of one or more procedures within a system maintaining positional encoding and the physical and positional encodings of the units may be associated. This association between physical encodings and products can be used in downstream procedures even in the case where the positional encoding of the units is lost, for example when the units have been removed from an ordered 1d-array or otherwise disordered with respect to one another.Pumps

[0194] The systems and devices described in further detail elsewhere herein may contain pumps, for example for moving solutions or units through the channels of microfluidic devices, or for delivery of reagents into the reaction chambers of microfluidic devices. These pumps may be mechanical or non-mechanical, and utilize driving forces such as piezoelectrical, electrostatic, electro-osmotic, solenoid, thermo-pneumatic, pneumatic, magnetic, vacuum, or passive gravity or capillary forces, or other appropriate forces known to those of skill in the art (see Iverson B D et al, 2008, incorporated herein by reference in its entirety). The pumps may comprise peristaltic, syringe, vacuum, piezoelectric, or passive, or other appropriate pumps known to those of skill in the art. The pump may be connected to a flow rate sensor and a pressure controller.

[0195] In various embodiments, pumps are used for routing (e.g. steering units through several junctions, e.g. Y-junctions, to destination(s), e.g. a microfluidic destination, such as intone or more reaction chambers. The pump described herein may be used to generate pulses of about 100 pl over <10 msec. In some embodiments, pumps described herein are configured to generate pulses of about, at least, or at least about 1 pl, 2 pl, 3 pl, 4 pl, 5 pl, 6 pl, 7 pl, 8 pl, 9 pl, 10 pl, 20 pl, 30 pl, 40 pl, 50 pl, 60 pl, 70 pl, 80 pl, 90 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 1 nl, 2 nl, 3 nl, 4 nl, 5 nl, 6 nl, 7 nl, 8 nl, 9 nl, 10 nl, or more. The pumps described herein may be configured to generate less than or less than about 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 1000 nl, 900 nl, 800 nl, 700 nl, 600 nl, 500 nl, 400 nl, 300 nl, 200 nl, 100 nl, 90 nl, 80 nl, 70 nl, 60 nl, 50 nl, 40 nl, 30 nl, 20 nl, 10 nl, 9 nl, 8 nl, 7 nl, 6 nl, 5 nl, 4 nl, 3 nl, 2 nl, 1 nl, 900 pl, 800 pl, 700 pl, 600 pl, 500 pl, 400 pl, 300 pl, 200 pl, 100 pl, 90 pl, 80 pl, 70 pl, 60 pl, 50 pl, 40 pl, 30 pl, 20 pl, 10 pl, 9 pl, 8 pl, 7 pl, 6 pl, 5 pl, 4 pl, 3 pl, 2 pl, 1 pl or less. Those of skill in the art will appreciate that the pumps described herein may be used to generate pulses of a volume that falls within any range bound by any of these values, for example 10-50 pl, 10-100 pl, 50-100 pl, 100-200 pl, 200-300 pl, 300-400 pl, 400-500 pl, 500-600 pl, 600-700 pl, 700-800 pl, 800-900 pl, 900-1000 pl, or 1-10 pl. Values for the pulse volumes generated by the pumps described herein may range between any of the potential values set forth for the pulse volumes herein. In some embodiments, pumps described herein are configured to generate pulses of volumes described herein in over about, less than or less than about 500 msec, 400 msec, 300 msec, 200 msec, 100 msec, 90 msec, 80 msec, 70 msec, 60 msec, 50 msec, 40 msec, 30 msec, 25 msec, 20 msec, 10 msec, 9 msec, 8 msec, 7 msec, 6 msec, 5 msec, 4 msec, 3 msec, 2 msec, 1 msec, 0.9 msec, 0.8 msec, 0.7 msec, 0.6 msec, 0.5 msec, 0.4 msec, 0.3 msec, 0.2 msec, 0.1 msec or less.

[0196] Any appropriate pump can be used, including, but not limited to, electroosmotic pumps, which are cost-effective, compact, and can achieve high flow rates with a sub-millisecond response time.

[0197] A pump may be used to route (e.g. steer) units, e.g. microbeads via cross-flow routing. In some embodiments, the units within the microfluidics devices described herein are passed through a primary channel, which branches into at least two side channels, e.g. in a Y-shaped geometry. At least two additional cross-flow channels may be configured to intersect the main channel near the junction. As a unit, e.g. passes the cross-flow channels, a pulse of fluid may be delivered perpendicular to the unit's velocity. This pulse may be used to cause the unit to be deflected to the side of the channel. Laminar flow within the microfluidic devices described herein may be used to enable the unit to continue its forward motion in the same relative position in the channel adopted after deflection. Such deflected units may be caused to deterministically enter the side channel towards which it was deflected. FIG. 33 provides an illustration of a junction with cross-flow routing. Pneumatic pumps were used to move bead position laterally within laminar flow as individual beads were flowed from the left channel into the junction, thereby moving the beads' position within cross-section of the laminar flow toward one of the two branch channels on the right. Beads that were moved upward within the cross-section of the laminar flow followed a path into the upper branch channel and beads that were moved downward within the cross-section of the laminar flow followed a path into the lower branch channel.

[0198] FIG. 34B provides and exemplar electroosmotic pump. Electroosmotic pump elements of suitable configurations (such as a cylindrical frit 5 mm long and 1 mm in diameter) may be used to produce high pressures. In various embodiments, electroosmotic pump elements comprise a “pancake” design, for example one comprising a frit 5 mm in diameter and 1 mm long. Suitable pump elements may be selected in order to improve ability to prime, (such as by decreasing resistance to flow; to increase flow rates; and / or to improve ease of assembly. Any suitable method known in the art may be used to fabricate such electroosmotic pump elements.

[0199] Also provided herein is a high-speed pump, such as an electroosmotic high-speed pump, capable of sorting beads into reaction chambers. In various embodiments, such pumps may be configured to route units, e.g. beads, at, at about, or at least 1 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 42 Hz, 45 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, 5 kHz, 6 kHz, 7 kHz, 8 kHz, 9 kHz, 10 kHz, or greater. Such pumps may be configured to achieve such routing frequencies with an accuracy of, of about, or of at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.99%, 99.993%, 99.995% 99.999%, 99.9995% 99.9999% or greater. In some embodiments, the pump is configured to have a flow rate of, of about, or of at least 25 nl / s and / or a switching rate of, of about, or of at least 30 Hz for at least 1 hour. In some embodiments, the pump is configured to have a flow rate of, of about, or of at least 10 nL / s and / or rise times (t) of, of about, or of at least 10 msec, 9 msec, 8 msec, 7 msec, 6 msec, 5 msec, 4 msec, 3 msec, 2 msec, 1 msec, 900 μsec, 800 μsec, 700 μsec, 600 μsec, 500 μsec, 400 μsec, 300 μsec, 200 μsec, 100 μsec, 50 μsec, 10 μsec, 5 μsec, or 1 μsec, or less when integrated into a fluidic system.

[0200] In some embodiments, microfluidic devices comprising such pumps may be configured to result the synthesis of, of about, or of at least 10,000 oligonucleotides. Such oligonucleotides may be, be about, or be at least 5, 10, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10,000 nucleotides long, or longer. In various embodiments, microfluidic devices comprising such pumps may be used to synthesize such oligonucleotides in, in about, or in less than 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours or less. Such microfluidic devices may be configured to achieve such oligonucleotide synthesis with, with about, or with fewer than 1000, 500, 400, 300, 200, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30 20, 10, 5, 4, 3, 2 or fewer errors (i.e. units, such as beads, with an incorrect sequence).

[0201] In some embodiments, the pump has at least a single microfluidic bifurcation and manual or semi-manual switched routing, e.g. steering, of, of about, or of at least 100 particles with, with about or with at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.99%, 99.995%, or 100% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 100 individual particles at, at about, or at at least 1 Hz through a single bifurcation with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995%, or 100% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 1000 individual particles at, at about or at at least 5 Hz through two serial bifurcations into four outlets with, with about, or with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995% accuracy. In some embodiments, the pump has automated routing, e.g. steering, of, of about, or of at least 1000, 5,000, 10,000, 50,000, 100,000, 500,000 or 1,000,000 individual particles at, at about or at least 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 200, 2500, 3000, 3500, 4000, 4500, or 5000 Hz through two serial bifurcations into at least one, two, or four outlets with, with about, or with at least 95%, 96%, 97%, 98%, 99% 99.5%, 99.9%, 99.95%, 99.99%, 99.995% accuracy.

[0202] In some embodiments, the pump is a piezo or solenoid driven pump.

[0203] The mobile units may be in a fluid or solution. Pumps may be used to control the flow rate and / or pressure of the fluid and thereby control the flow rate of the units. Pumps may also be used to control the direction of the fluid or solution flow in the device and thereby control the flow direction of the unit. Changes in the flow direction of a fluid may be used to distribute the mobile units into secondary channels, branch channels, branch points, or reaction chambers. For example, a pump at the first end of a channel may apply a flow rate such that the units move down the channel to a branch point that branches into two, three, or more channels. The branch point may comprise a router, e.g. a distributor, or may not comprise a router. As the units approach the branch point, the pump at the first end of the channel is shut off or slowed, and a second pump at the end of one of the branch channels is turned on, resulting in flow of the fluid comprising the mobile units towards the second pump and down the chosen branch channel. Each branch channel may have a separate pump that can be controlled independently. Mobile units can be routed into the individual branch channels by turning on the appropriate pump for each branch channel as the unit approaches or passes through the branch point. Individual units or groups of units may be routed into branch channels.

[0204] Units in a fluid may be passed through the channels or the path of a detector at a flow rate of about, at least, or at least about 10 nl / min, 20 nl / min, 30 nl / min, 40 nl / min, 50 nl / min, 60 nl / min, 70 nl / min, 80 nl / min, 90 nl / min, 100 nl / min, 200 nl / min, 300 nl / min, 400 nl / min, 500 nl / min, 600 nl / min, 700 nl / min, 800 nl / min, 900 nl / min, 1 μl / min, 2 μl / min, 3 pl / min, 4 μl / min, 5 μl / min, 6 μl / min, 7 μl / min, 8 μl / min, 9 μl / min, 10 μl / min, 20 μl / min, 30 pl / min, 40 μl / min, 50 μl / min, 60 μl / min, 70 μl / min, 80 μl / min, 90 μl / min, 100 μl / min, or faster. In some cases, units in a fluid may be passed through the path of a detector at a flow rate of at most, or at most about 100 μl / min, 90 μl / min, 80 μl / min, 70 μl / min, 60 μl / min, 50 pl / min, 40 μl / min, 30 μl / min, 20 μl / min, 10 μl / min, 9 μl / min, 8 μl / min, 7 μl / min, 6 μl / min, 5 pl / min, 4 μl / min, 3 μl / min, 2 μl / min, 1 μl / min, 100 nl / min, 90 nl / min, 80 nl / min, 70 nl / min, 60 nl / min, 50 nl / min, 40 nl / min, 30 nl / min, 20 nl / min, 10 nl / min, or slower. Those of skill in the art appreciate that the flow rate may fall within any range bound by any of these values, for example 10-100 nl / min, 100-500 nl / min, or 500-1000 nl / min. Units and / or carrier fluid may also be passed through the device at a flow rate of about, at least, or at least about 0.1 cm / min, 0.5 cm / min, 1 cm / min, 2 cm / min, 3 cm / min, 4 cm / min, 5 cm / min, 6 cm / min, 7 cm / min, 8 cm / min, 9 cm / min, 10 cm / min, 20 cm / min, 30 cm / min, 40 cm / min, 50 cm / min, 60 cm / min, 70 cm / min, 80 cm / min, 90 cm / min, 1 m / min, 2 m / min, 3 m / min, 4 m / min, 5 m / min, 6 m / min, 7 m / min, 8 m / min, 9 m / min, 10 m / min, 20 m / min, 30 m / min, 40 m / min, 50 m / min, 60 m / min, 70 m / min, 80 m / min, 90 m / min, 100 m / min, or faster. In some cases, carrier fluid and / or units in a fluid may be passed through the channels or the path of a detector at a flow rate of at most, or at most about 100 m / min, 90 m / min, 80 m / min, 70 m / min, 60 m / min, 50 m / min, 40 m / min, 30 m / min, 20 m / min, 10 m / min, 9 m / min, 8 m / min, 7 m / min, 6 m / min, 5 m / min, 4 m / min, 3 m / min, 2 m / min, 1 m / min, 90 cm / min, 80 cm / min, 70 cm / min, 60 cm / min, 50 cm / min, 40 cm / min, 30 cm / min, 20 cm / min, 10 cm / min, 9 cm / min, 8 cm / min, 7 cm / min, 6 cm / min, 5 cm / min, 4 cm / min, 3 cm / min, 2 cm / min, 1 cm / min, 0.5 cm / min, 0.1 cm / min, or slower. Those of skill in the art appreciate that the carrier fluid and / or flow rate may fall within any range bound by any of these values, for example 10-100 cm / min, 100-500 cm / min, or 500-1000 cm / min. Values for the flow rate may range between any of the potential values set forth for the flow rate herein.

[0205] In various embodiments, pumps may be used to facilitate movement of mobile units. A pump may be attached to a channel to manipulate the flow rate of the fluid in the channel. The flow can be stopped, started, or the flow rate modulate via the speed of the pump, resulting in stopping, starting, or modulation of the unit movement through the device. Pump-controlled fluid flow may also be used to route, e.g. distribute, the mobile units by creating low pressure or vacuum conditions in the desired direction of travel for the mobile unit.

[0206] The methods and compositions described herein may be used to order units within a microfluidic device. Any suitable type of distributing algorithm can be used to distribute units in a first order into a second order. For example, units in a device may be distributed so that the correct units could be dispensed at the correct time or order. A first group of units may be dispensed followed by a second group of units and so on. In some embodiments, the exact order of the units within each such group is unimportant. Accordingly, units may be distributed so that the correct units are grouped into a first group of a desired size, a second group of a desired size etc. For example, the first group in a given grouping may have a size of 5 units whereas the second group in the grouping may have a size of 1 unit.Valves and Bead Stops

[0207] The device may contain elastomeric valves that close off sections of the channel(s). These valves may be mechanical or pressure-actuated. The valves may be deflected into or retracted from one channel or channel section in response to a force applied to another channel or channel section. The valves may be upwardly-deflecting, downwardly deflecting, side actuated, normally-closed, or some other type of valve. Elastomeric valves for use in microfluidic devices are described in US 20050072946, U.S. Pat. No. 6,408,878, US 20020127736, and U.S. Pat. No. 6,899,137, all which are herein incorporated by reference in their entirety, in particular with respect to the description of elastomeric valves. The device may have a combination of valve types. The valves may be operated by injecting gases, liquids, ionic solutions, or polymer solutions. A non-exclusive list of such solutions includes air, nitrogen, argon, water, silicon oils, perfluoropolyalkylether or other oils, salt solutions, polyethylene glycol, glycerol, and carbohydrates. Valves may also be operated by applying a vacuum to the channel(s).

[0208] The device may also contain valves that are physically separated from the reaction chamber(s) and / or branch channel(s). Reagents may be routed to the reaction chamber(s) and / or branch channel(s) via a delivery channel or an inlet directly or indirectly via a network of channels. In some embodiments, the delivery channel and / or inlet is about the same size or smaller than the reaction chamber(s), branch channel(s), and / or other channel(s) connecting the delivery channel and / or inlet to where reagents are designated to be delivered. In some embodiments, a delivery channel and / or an inlet interfaces with the reaction chamber(s), branch channel(s), and / or other connected channel(s) via a frit, a nozzle, a weir, a bead stop, or any other physical structure that enable fluid to pass through the structure but not units.

[0209] Valves and valve membranes can be constructed from any appropriate elastomeric material known in the art, including poly dimethylsiloxane (PDMS), polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicones. A non-exclusive list of elastomeric materials which may be utilized in connection with the present invention includes polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicone polymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F), perfluoropolyalkylether siloxane block copolymer, poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(1-butene), poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton), elastomeric compositions of polyvinylchloride (PVC), polysulfone, polycarbonate, polymethylmethacrylate (PMMA), and polytertrafluoro-ethylene (Teflon).

[0210] In some embodiments, the device includes one or more microfluidic check valves. A microfluidic check valve can be used to direct solution flow in only one direction through the valve. Any suitable check valve known in the art may be used in the systems and devices described herein.

[0211] Valve membranes separating flow channels may have a thickness of between about 0.01 and 1000 microns. Membrane thicknesses can be about, at least, or at least about 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm 7 μm, 8 μm, 9 μm 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm. Membrane thicknesses can be less than or less than about 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, 0.09 μm, 0.08 μm, 0.07 μm, 0.06 μm, 0.05 μm, 0.04 μm, 0.03 μm, 0.02 μm, 0.01 μm. Those of skill in the art will appreciate that the membrane thickness may have a size that falls within any range bound by any of these values, for example 0.01-0.1 μm, 0.1-1 μm, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm. Values for the valve membrane thickness may range between any of the potential values set forth for the valve membrane thickness herein.

[0212] In some embodiments, the device described herein includes unit stops, such as a frit, wire, or weir. Unit stops may be used to halt the flow of single or multiple mobile units in one direction. Any appropriate unit stop known in the art may be used. Unit stops may be manufactured by inserting a wire within a channel, 3D printing a capillary connector that introduces a constriction or frit, and / or using photolithography to create a weir structure in a glass device or any suitable method known in the art. Unit stops may be used to halt the flow of single or multiple mobile units in one direction. The stopped mobile units may then be held, or the flow of the stopped mobile units may be reversed by altering the fluid flow or pressure e.g. via a pressure controller, pump, or vacuum. Unit stops may be used at any point in the device, such as at the beginning or end of a channel or branch channel, at a branch point, at the beginning or end of a reaction chamber, or any combination thereof.Detectors and Optical Detection Systems

[0213] The microfluidic devices described in various embodiments herein may include one or more detection systems for positionally tracking units within the microfluidic device. Each detection system may have one or more detectors. One or more detectors may be placed at any point in the device, for example to track units in a channel or the device, such as at any point in a channel or branch channel, before or after any or every branch point, before or after any or every router, e.g. distributor, before or after any or every reaction chamber, or before or after any or every outlet or inlet. One or more detectors may be used to ensure the correct number of units are distributed or steered into a channel or branch channel. In various embodiments, the one or more detectors are not be restricted to particular points or junctions of the systems described herein. For instance, the one or more detectors can be configured globally or regionally relative to a particular system. In one such embodiment, a charge-coupled device (CCD) / complementary metal oxide semiconductor (CMOS) or other wide-field imaging detector could monitor some or all of the units in a fluidic network of the system, or could be used to observe a region-of-interest (ROI) (e.g. to improve detection speed or reduce data volume). Additionally or alternatively, linear CCD arrays can be used to track beads in multiple channels (e.g., parallel channels, non-parallel channels). Additionally or alternatively, regional detectors, including but not limited to CCDs, can incorporate machine vision to identify and track beads as they move through the device. Additionally or alternatively, conductivity-based detection may be used to identify the presence and / or quantity of units, e.g. beads along a fluidic path between electrode probe points. In any embodiment, detectors may also be configured in any orientation relative to the channel(s) of the microfluidic devices described herein (e.g., above, below, laterally).

[0214] A detection system may be configured to execute steps for serial or parallel interrogation of the units using a variety of interrogatory devices, such as interrogatory devices using lasers or cameras, real time classification, and rapid, command driven distributing. The detection system may comprise a multiple part system, having, for example, one or more of a scanner that emits light at a particular excitation wavelength or set of wavelengths over the units in the microfluidic device, a detector that receives the emitted light or diffraction pattern from the units and converts it into a digital electrical signal that corresponds to the unit, a decoder that translates the signal into data which can then be sent to an associated computer for storage, and / or any other suitable component known in the art. Light illumination and detection devices may include fluorescence, surface plasmon resonance, total internal reflection fluorescence (TIRF), Raman spectroscopy, or any other suitable light illumination and / or detection technique known in the art. Detectors may include non-optical detectors such as magnetic detectors, conductivity sensors such as Coulter counters, capacitive sensors, dielectric spectroscopy, or any other non-optical detector known in the art, or any combination thereof. Multiple detectors, and multiple types or classes of detectors may be used in the device as described herein. For example, a device may have both one or more optical detectors and one or more non-optical detectors.

[0215] The detector may comprise a lamp (e.g. mercury, xenon, halogen), a laser (e.g. argon, krypton, helium neon, helium cadmium, diode laser), a light emitting diode (LED) or a diode laser coupled to a wavelength filter and a photon detector. The detector may also include a photomultiplier tube, a photodiode, or an avalanche photodiode. The detector may be optical fiber coupled or free-space optics coupled. The detector may also be a charge-coupled device (CCD) camera. Multiple detectors can be joined consecutively to read units that have multiple labels or to track a given unit through a device. Detectors configured to interrogate various locations within a device may collect information in parallel or in series.

[0216] Optical and non-optical detectors may detect and evaluate size, shape, orientation, positions, color, color spectra, interference patterns, barcode patterns, charge, magnetic or paramagnetic labels, or capacitance or conductivity of the units, or any combination thereof. Detectors may distinguish units from other non-unit elements such as dust, bubbles, unit fragments, or other contaminants. Detectors may be configured to collect location and speed information of units, which may be used for feedback control for the operation of the devices described herein, such as by increasing or decreasing the pressure of a carrier fluid to move the units, or to distribute the units. Detectors may be located in any channel, including without limitation a main channel, feeder channel, branch channel, reaction chamber or outlet channel and may be used to verify correct distributing or steering of the units, for example by determining the presence or absence of a unit, or by counting units to determine whether the correct number of units have been distributed or steered. Information collected by a detector may be used to identify an error in distributing and / or correct the distribution of a unit into the incorrect channel, as described in further detail elsewhere herein. As an example, a mis-distributed unit may be re-distributed into the correct channel, or a unit may be distributed into a channel to be held until it can be distributed into the correct channel.

[0217] An exemplary detector may comprise a single-mode or multimode source fiber and a receiver fiber placed adjacent or nearly adjacent to a channel. Such a detector is shown in FIG. 18. The source fiber provides an incident light and a receiver fiber receives light scattered or directed from the source fiber.

[0218] Highly accurate detection and counting of units can be achieved by using a detection system, such as an optical system to distinguish single units, even if closely spaced, from adjacent two (doubles), three (triplets), or more units (n-tuplets) as they traverse the detection system in the device. Two adjacent units (a double) can be distinguished from one or more units through a characteristic detection patterns, for example a detection pattern comprising a characteristic light transmission pattern as shown in FIG. 16A. Single, double, triple, and n-tuple units are shown to each result in a different characteristic signal shape that can be used to distinguish the number of units or beads passing through the detector. Detecting these characteristic patterns allows for positionally tracking the units or beads as they move through the device.

[0219] Complex combinations of single, double, triple, and n-tuple units can be distinguished by a detection system, including, without limitation, an optical detection system. Optical detection systems described herein may be used to analyze signal patterns of transmitted light, as shown in shown in FIG. 16A as units pass through a detection path as described previously or elsewhere herein. Single, double, triple, and n-tuple units traversing through an optical detection system may be identified by a characteristic intensity signal signature, including, without limitation, the characteristic “W” pattern obtained by single beads passing through the optical detection system described in Example 4. Characteristic signal patterns for two or more adjacent units may be established using the detection systems described herein. Signal patterns may be used to distinguish single, double, triple, and n-tuple units, including for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more adjacent units. Values for the adjacent units may range between any of the potential values set forth for the adjacent units herein. Detectors described herein may be used to detect and / or count units in a stacked configuration or unstacked configuration and can be used to count an arbitrarily large number of units. Based on the identification of a plurality of adjacent units, systems and methods described herein may be used to take an action on the plurality of adjacent beads. Such an action may include corrective mechanisms, including without limitation, directing one or more units, such as one or more of the detected adjacent units, to a holding chamber, applying a separating force on one or more units, such as one or more of the detected adjacent units, reprogramming of downstream directions of one or more units, such as one or more of the detected adjacent units, or combinations of one or more of the foregoing. One or more units, such as one or more beads that miss the application of designated application of reaction conditions, may be redirected immediately or at a later point such that the missed application of reaction conditions can be applied.

[0220] Without being bound by theory, the optical detection signals may be generated by the incident light being scattered (FIGS. 17A and 17B), and transmission intensity decreasing from baseline intensity as the leading edge of a first unit enters the optical path of an optical detection system (a) (FIG. 17B). Then, as the center of the first unit aligns with the optical path, the transmitted light momentarily increases, likely due to lensing of some, but not all of the light through the unit and into the receiving fiber (b). Transmitted light intensity decreases even further as the trailing edge of the first unit and leading edge of the second bead directly align with the optical path (c). Then, the transmitted light momentarily increases again, likely due to lensing as the center of the second unit aligns with the optical path (d). Transmitted light then momentarily decreases one last time as the trailing edge of the second unit traverses the optical path (e). This results in a characteristic “W” shape of the signal.

[0221] In various embodiments, methods and systems described herein are configured to distinguish bubbles from units in order to detect bubbles within the microfluidic devices described herein. Without being bound by theory, bubbles may interfere with device operation and / or cause miscounting of units. Bubbles traveling through a detector, such as an optical path lens, may cause a similar signal at the detector as that of a unit, for example a bead. In various embodiments, detectors, including without limitation, the optical detection systems described herein may be designed to distinguish bubbles from units using various characteristics. For example, bubbles may have a lower index of refraction than units, for example beads. The use of a sufficiently sensitive optical sensing system allows discrimination between the change in signal intensity from baseline caused by a bubble from that caused by a unit, for example a bead. In addition, a narrow size distribution of the units within the systems described herein reduces variation in unit signals, including for example the variation in signal width of a unit passing through the path of a detector at a selected speed. Without being bound by theory, greater bubble size variation can cause a greater variation in bubble signals. The combination of signal width variation and signal intensity differences can be combined to discriminate bubbles from other types of units in methods and systems described herein (FIGS. 18A and 18B).

[0222] Detectors may be configured to collect information from units passing the detector's path at a rate of about, at least, or at least about 1×10−1 units / sec (u / sec), 1×101 u / sec, 1×102 u / sec, 2×102 u / sec, 3×102 u / sec, 4×102 u / sec, 5×102 u / sec, 6×102 u / sec, 7×102 u / sec, 8×102 u / sec, 9×102 u / sec, 1×103 u / sec, 2×103 u / sec, 3×103 u / sec, 4×103 u / sec, 5×103 u / sec, 6×103 u / sec, 7×103 u / sec, 8×103 u / sec, 9×103 u / sec, 1×104 u / sec, 2×104 u / sec, 3×104 u / sec, 4×104 u / sec, 5×104 u / sec, 6×104 u / sec, 7×104 u / sec, 8×104 u / sec, 9×104 u / sec, 1×105 u / sec, 2×105 u / sec, 3×105 u / sec, 4×105 u / sec, 5×105 u / sec, 6×105 u / sec, 7×105 u / sec, 8×105 u / sec, 9×105 u / sec, 1×106 u / sec, 2×106 u / sec, 3×106 u / sec, 4×106 u / sec, 5×106 u / sec, 6×106 u / sec, 7×106 u / sec, 8×106 u / sec, 9×106 u / sec, 1×107 u / sec, 2×107 u / sec, 3×107 u / sec, 4×107 u / sec, 5×107 u / sec, or more. In some cases, detectors may be configured to collect information from units passing through the detector's path at a rate of at most, or at most about 5×107 u / sec, 4×107 u / sec, 3×107 u / sec, 2×107 u / sec, 1×107 u / sec, 9×106 u / sec, 8×106 u / sec, 7×106 u / sec, 6×106 u / sec, 5×106 u / sec, 4×106 u / sec, 3×106 u / sec, 2×106 u / sec, 1×106 u / sec, 9×105 u / sec, 8×105 u / sec, 7×105 u / sec, 6×105 u / sec, 5×105 u / sec, 4×105 u / sec, 3×105 u / sec, 2×105 u / sec, 1×105 u / sec, 9×104 u / sec, 8×104 u / sec, 7×104 u / sec, 6×104 u / sec, 5×104 u / sec, 4×104 u / sec, 3×104 u / sec, 2×104 u / sec, 1×104 u / sec, 9×103 u / sec, 8×103 u / sec, 7×103 u / sec, 6×103 u / sec, 5×103 u / sec, 4×103 u / sec, 3×103 u / sec, 2×103 u / sec, 1×103 u / sec, 9×102 u / sec, 8×102 u / sec, 7×102 u / sec, 6×102 u / sec, 5×102 u / sec, 4×102 u / sec, 3×102 u / sec, 2×102 u / sec, 1×102 u / sec, 1×101 u / sec, 1×10−1 u / sec or less. Those of skill in the art appreciate that the unit passing rate may fall within any range bound by any of these values, for example 1×102-1×103 u / sec, 1×103-5×103 u / sec, or 5×103-1×104 u / sec. Values for the information collection rate may range between any of the potential values set forth for the information collection rate herein.Nucleic Acid Synthesis

[0223] In one embodiment, the synthesis of large library of specific DNA or other nucleic acid molecules is achieved according to the methods and compositions described herein. A set of units begin in a primary channel and are directed according to a preassigned program to one of four distinct channels. Direction into these channels may be achieved by a multiway distributor, by two sequential bifurcations and corresponding two-way distributors, or by any other suitable method known in the art. Reagents, such as various phosphoramidites may be delivered to the channels. The units may be combined maintaining their positional encoding and reassigned and delivered into one of the four distinct channels. Accordingly, nucleotides may be added in iterative steps to a nascent chain on each unit.

[0224] In various embodiments, nucleic acid synthesis is performed in or on the units described herein within the microfluidic devices described herein. In some cases, nucleic acid synthesis is achieved using the phosphoramidite method. Alternative nucleic acid synthesis methods may also be used, such as H-phosphonate, phosphate triester, phosphodiester, phosphotriester, and phosphite triester methods. A non-exclusive list of reagents for these methods that may be delivered to the units comprises nucleotide phosphoramidite monomers; non-nucleoside phosphoramidite monomers; B-cyanoethyl; 4,4′-dimethoxytrityl (DMT); tricholroacetic acid and / or dochloroacetic acid; an acedic azole catalyst, such as 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or other similar compounds; acetic anhydride, 1-methylimidazole, and / or DMAP; iodine; water; a weak base such as pyridine, lutidine, or collidine; tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO); 3-(Dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, and / or N,N,N′N′-Tetraethylthiuram disulfide; and controlled porous glass. Reagents for nucleic acid synthesis are available from purchase from numerous commercial sources, including American International Chemical (Natick Mass.), BD Biosciences (Palo Alto Calif.), and others. The specific reagents used may vary depending on the method of nucleic acid synthesis, e.g phosphoramidite or non-phosphoramidite reactions.

[0225] In some embodiments, nucleotides with suitable modifications for phosphoramidite or non-phosphoramidite chemistry are deposited on a functionalized unit(s) in the device. These nucleotides can be mononucleotides, dinucleotides, or longer oligonucleotides. Phosphoramidite-based nucleic acid synthesis chemistry typically involves the following steps in order: 1) coupling, 2) capping, 3) oxidation and / or sulfurization, 4) deblocking, and 5) desalting. Either oxidation or sulfurization may be used as one of the steps. Successive rounds of chemistry performed in the device may result in step-wise synthesis of high-quality polymers on units. In various embodiments, units described herein are subjected to one or more steps of nucleic acid synthesis in the microfluidic devices described herein. For example, one or more units in a reaction chamber may be contacted with reagents and solutions through one or more reagent channels that connect to the reaction chamber.

[0226] Methods of quickly synthesizing n-mer, such as about or at least about 100-, 150-, 200, 250-, 300, 350-, or longer nucleotide, oligonucleotides on units is further described herein in various embodiments. Such methods can use units functionalized with a chemical moiety suitable for nucleotide coupling. In various embodiments, the surface of the units described herein is chemically modified to provide a proper site for the linkage of a growing oligomer to the surface.

[0227] In some embodiments, a trialkoxysilyl amine (e.g. (CH3CH20)3Si—(CH2)2-NH2) is allowed to react with glass or silica surface SiOH groups, followed by reaction with succinic anhydride with the amine to create an amide linkage and a free OH on which the nucleotide chain can grow. In some embodiments, beads, such as polymeric beads e.g. polystyrene beads or divinylbenzene-cross-linked polystyrene beads comprise amino functionalization, hydroxyl functionalization, or other suitable functionalization known in the art.

[0228] In some embodiments, photocleavable linkers are used. A photocleavable linker may allow for a synthesized oligonucleotide to be removed from the units (e.g. by irradiation with light, e.g. ˜350 nm light) without cleaving the protecting groups on the nitrogenous functionalities on each base. The use of photocleavable linkers of this sort is described at www.glenresearch.com. Various other suitable cleavable linker groups are known in the art and may alternatively be used.

[0229] In some embodiments, adenine, guanine, thymine, cytosine, or uridine building blocks, or analogs / modified versions thereof are used as described in further detail elsewhere herein. In some cases, the added building blocks comprise dinucleotides, trinucleotides, or longer nucleotide based building blocks, such as building blocks containing about or at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. In some embodiments, large libraries of n-mer oligonucleotides are synthesized in parallel on units, e.g. about or at least 100, 1000, 10000, 100000, 1000000, 2000000, 3000000, 4000000, 5000000 units hosting oligonucleotide synthesis. Individual units may host synthesis of oligonucleotides that are different from each other.

[0230] A common method for the preparation of synthetic nucleic acids is based on the fundamental work of Caruthers and is known as the phosphoramidite method (M. H. Caruthers, Methods in Enzymology 154, 287-313, 1987; incorporated herein by reference in its entirety).

[0231] In some embodiments, the synthesis of DNA oligomers by the methods of the invention is achieved through phosphoramidite chemistry, reviewed in Streyer, Biochemistry (1988) pp 123-124 and U.S. Pat. No. 4,415,732, herein incorporated by reference. In various embodiments, the chemical synthesis of nucleic acids is performed using variations of the phosphoramidite chemistry developed for solid surfaces (Beaucage S L, Caruthers M H. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 1981; 22: 1859-1862; Caruthers M H. Gene synthesis machines—DNA chemistry and its uses. Science. 1985; 230:281-285., both of which are incorporated herein by reference in their entirety). For instance, phosphoramidite based methods can be used to synthesize abundant base, backbone and sugar modifications of deoxyribo- and ribonucleic acids, as well as nucleic acid analogs (Beaucage S L, Iyer R P. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron. 1992; 48:2223-2311; Beigelman L, Matulic-Adamic J, Karpeisky A, Haeberli P, Sweedler D. Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies. Methods Enzymol. 2000; 317:39-65; Chen X, Dudgeon N, Shen L, Wang J H. Chemical modification of gene silencing oligonucleotides for drug discovery and development. Drug Discov. Today. 2005; 10:587-593; Pankiewicz K W. Fluorinated nucleosides. Carbohydrate Res. 2000; 327:87-105; Lesnikowski Z J, Shi J, Schinazi R F. Nucleic acids and nucleosides containing carboranes. J. Organometallic Chem. 1999; 581: 156-169; Foldesi A, Trifonova A, Kundu M K, Chattopadhyaya J. The synthesis of deuterionucleosides. Nucleosides Nucleotides Nucleic Acids. 2000; 19: 1615-1656; Leumann C J. DNA Analogues: from supramolecular principles to biological properties. Bioorg. Med. Chem. 2002; 10:841-854; Petersen M, Wengel J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 2003; 21:74-81; De Mesmaeker A, Altmann K-H, Waldner A, Wendebom S. Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr. Opin. Struct. Biol. 1995; 5:343-355), all of which are incorporated herein by reference in their entirety.

[0232] In various embodiments, nucleic acids, e.g. nucleic acid double strands, are synthesized and / or assembled while attached on a unit. Methods of preparation of nucleic acid on a common solid support, are discussed in U.S. Pat. No. 7,790,369 and Pub. No. 2007-0087349, both of which are herein incorporated by reference in their entirety.Oligonucleotides

[0233] As used herein, the terms “preselected sequence”, “predefined sequence” or “predetermined sequence” are used interchangeably. The terms mean that the sequence of the polymer is known and chosen before synthesis or assembly of the polymer. Various aspects of the invention are described herein primarily with regard to the preparation of nucleic acids molecules, the sequence of the oligonucleotide or polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules. In one embodiment, oligonucleotides are short nucleic acid molecules. For example, oligonucleotides may be from about 10 to about 300 nucleotides, from about 20 to about 400 nucleotides, from about 30 to about 500 nucleotides, from about 40 to about 600 nucleotides, or more than about 600 nucleotides long. Those of skill in the art appreciate that the oligonucleotide lengths may fall within any range bounded by any of these values (e.g., from about 10 to about 400 nucleotides or from about 300 to about 400 nucleotides etc.). Suitably short or long oligonucleotides may be used as necessitated by the specific application. Individual oligonucleotides may be designed to have a different length from another in a library. Oligonucleotides can be relatively short, e.g. shorter than 200, 100, 80, 60, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, or 4 nucleotides, more particularly. Relatively longer oligonucleotides are also contemplated; in some embodiments, oligonucleotides are longer than or equal to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 500, 600 nucleotides, or longer. In some embodiments, oligonucleotides are single-stranded DNA or RNA molecules.

[0234] In some embodiments, oligonucleotides described herein are associated to the units described herein through an attachment via the oligonucleotides' 5′ end. In some embodiments, oligonucleotides described herein are associated to the units described herein through an attachment via the oligonucleotides' 3′ end. In some embodiments, the attachment is through a covalent bond or an affinity binding pair. In some embodiments, a second strand complementary to a first strand associated with a unit is synthesized. In some embodiments, the second strand is attached to the unit via its 3′ end. In some embodiments, the second strand is attached to the unit via its 5′ end. In some embodiments, oligonucleotides associated with a unit have a free 3′ end. In some embodiments, oligonucleotides associated with a unit have a free 5′ end. Oligonucleotides described under this paragraph may be synthesized using any synthesis method described herein or any suitable method known in the art.

[0235] In some embodiments, oligonucleotides attached to a unit are inverted to reverse the orientation with respect to the units, e.g. from 3′-bound to 5′-bound. Oligonucleotides described herein may be attached to units described herein through a cleavable linker, e.g. a cleavable linker arm of a branched linker attached to a unit. In various embodiments, a branched linker is attached to units described herein. Such branched linkers may comprise a first branch, e.g. a first branch comprising a first alkyne and a second branch, e.g. a second branch comprising a cleavable linker. An oligonucleotide may be attached to the cleavable linker via a first end, e.g. via its 3′ end. The other end, e.g. the 5′ end, of the oligonucleotide, may be attached to an azide group. In some embodiments, the oligonucleotide is circularized through the branched linker, e.g. by binding the free end of the oligonucleotide to the second branch. In some embodiments, the circularization is achieved by Cu(I) Click chemistry. For example, the azide group on the free end of the oligonucleotide may be reacted with the alkyne on the first branch of the branched linker. Upon circularization, the cleavable linker may be cleaved, e.g. using standard deprotection conditions, such as treatment with NH4OH at 55° C. for 15 hours, thereby releasing the first end, e.g. 3′ end of the oligonucleotide. Methods relating to inversion of oligonucleotides on solid supports are further discussed in U.S. Pat. Pub. 2017 / 0050162, which is incorporated herein by reference in its entirety.

[0236] In one aspect of the invention, a device for synthesizing a plurality of nucleic acids having a predetermined sequence is provided. The device can include units as described in further detail herein having a plurality of oligonucleotides. In some embodiments, the oligonucleotides are linked through their 3′ end to the units described herein. Yet, in other embodiments the oligonucleotides are linked through their 5′ end to the units described herein. Oligonucleotide linkages may be in a variety of forms, such covalent linkages or linkages comprising affinity binding.

[0237] An oligonucleotide may be immobilized on the units described herein via a nucleotide sequence (e.g., a degenerate binding sequence), a linker or spacer (e.g., a moiety that is not involved in hybridization). In some embodiments, the oligonucleotide comprises a spacer or linker to separate the oligonucleotide sequence from the unit. Useful spacers or linkers include photocleavable linkers, or other traditional chemical linkers. In one embodiment, oligonucleotides may be attached to a unit through a cleavable linkage moiety. For example, the unit may be functionalized to provide cleavable linkers for covalent attachment to the oligonucleotides. The linker moiety may be of six or more atoms in length. Alternatively, the cleavable moiety may be within an oligonucleotide and may be introduced during synthesis. A broad variety of cleavable moieties are available in the art of oligonucleotide synthesis (see e.g., Pon, R., Methods Mol. Biol. 20:465-496 (1993); Verma et al, Annu. Rev. Biochem. 67:99-134 (1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728). A suitable cleavable moiety may be selected to be compatible with the nature of the protecting group of the nucleoside bases, the choice of unit material, and / or the mode of reagent delivery, among others. In an exemplary embodiment, the oligonucleotides cleaved from the unit contain a free 3′-OH end. Alternatively, the free 3′-OH end may also be obtained by chemical or enzymatic treatment, following the cleavage of oligonucleotides. In various embodiments, the invention relates to methods and compositions for release of unit bound oligonucleotides into solution. The cleavable moiety may be removed under conditions which do not degrade the oligonucleotides. The linker may be cleaved using a variety of approaches, for example simultaneously under the same conditions as a deprotection step or subsequently utilizing a different condition or reagent for linker cleavage after the completion of a deprotection step.

[0238] As used herein, the term “duplex” may refer to a nucleic acid molecule that is at least partially double-stranded. The terms “nucleoside” or “nucleotide” may refer to those moieties which contain not only the conventional purine and pyrimidine bases, i.e., adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U), but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles or any other suitable modifications described herein or otherwise known in the art. In addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0239] As used herein, the terms “nucleoside” and “nucleotide” may refer to protected forms of nucleosides and nucleotides, e.g., wherein the base is protected with a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl, and purine and pyrimidine analogs.

[0240] Suitable nucleotide and nucleoside analogs will be known to those skilled in the art. Common analogs include, but are not limited to, 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methyl guanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylanminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxy acetic acid, uracil-5-oxy acetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine.

[0241] As used herein, the term “oligonucleotide” shall include poly deoxynucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones (for example PNAs). Thus, these terms include known types of oligonucleotide modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). There is no intended distinction in length between the term “polynucleotide” and “oligonucleotide,” and these terms may be used interchangeably.

[0242] The term “attached,” as in, for example, a unit having a moiety “attached” thereto, includes covalent binding, adsorption, and physical immobilization. The terms “binding” and “bound” are identical in meaning to the term “attached.

[0243] Oligonucleotides on units may be cleaved from their solid surface and optionally pooled to enable new applications such as, gene assembly, nucleic acid amplification, sequencing libraries, shRNA libraries etc. (Cleary M A, Kilian K, Wang Y Q, Bradshaw J, Cavet G, Ge W, Kulkami A, Paddison P J, Chang K, Sheth N, et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nature Methods. 2004; 1:241-248), gene synthesis (Richmond K E, Li M H, Rodesch M J, Patel M, Lowe A M, Kim C, Chu L L, Venkataramaian N, Flickinger S F, Kaysen J, et al. Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res. 2004; 32:5011-5018; Tian J D, Gong H, Sheng N J, Zhou X C, Gulari E, Gao X L, Church G. Accurate multiplex gene synthesis from programmable DNA microchips. Nature. 2004; 432: 1050-1054) and site-directed mutagenesis (Saboulard D, Dugas V, Jaber M, Broutin J, Souteyrand E, Sylvestre J, Delcourt M. High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips. BioTechniques. 2005; 39:363-368), all of which are herein incorporated by reference in their entirety. In some embodiments, reactions comprising such oligonucleotides may be performed without detaching the oligonucleotide from its unit.”Other Oligomers

[0244] In various embodiments, the invention relates to the synthesis, such as chemical synthesis, of molecules other than nucleic acids. The terms “peptide,”“peptidyl” and “peptidic” as used throughout the specification and claims are intended to include any structure comprised of two or more amino acids. The peptides synthesized according to the methods described herein may comprise about 5 to 10,000 amino acids, preferably about 5 to 1000 amino acids. The peptides synthesized according to the methods described herein may comprise, comprise about or comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 amino acids or more. The peptides synthesized according to the methods described herein may comprise a number of amino acids falling within the range bounded by any of the foregoing values, such as 2-900, 90-10,000, etc. The amino acids forming all or a part of a peptide may comprise any of the twenty conventional, naturally occurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y) or amino acids, e.g. non-naturally occurring amino acids. The term “non-conventional amino acid” refers to amino acids other than conventional amino acids, and includes, for example, isomers and modifications of the conventional amino acids (e.g., D-amino acids), non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine), and peptides having the naturally occurring amide —CONH— linkage replaced at one or more sites within the peptide backbone with a non-conventional linkage such as N-substituted amide, ester, thioamide, retropeptide (—NHCO—), retrothioamide (—NHCS—), sulfonamido (—S02NH—), and / or peptoid (N-substituted glycine) linkages. Accordingly, the peptidic molecules of the array include pseudopeptides and peptidomimetics. The peptides of this invention can be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.

[0245] The term “oligomer” may encompass any polynucleotide or polypeptide or other chemical compound with repeating moieties such as nucleotides, amino acids, carbohydrates and the like.

[0246] In some examples, all or some of the units in a group of units, e.g. units within a microfluidic device described herein each are attached to a different composition, such as a different oligonucleotide.Amplification of Nucleic Acids

[0247] In various embodiments, the methods and systems relate to amplification of single stranded nucleic acids.

[0248] The single stranded nucleic acids may be amplified using adaptors incorporated into the target sequence. Polymerase chain reaction in conjunction with primers corresponding to these adaptors, or any amplification method described herein or any other suitable amplification method known in the art, can be used to amplify the target.

[0249] The single stranded nucleic acids may be circularized upon hybridization with an adaptor. A single stranded nucleic acid may be circularized by joining its 5′ and 3′ ends, forming a contiguous circle. Various ligation methods and enzymes are suitable for the reaction as described elsewhere herein and otherwise known in the art. Circularized nucleic acids may be attached to units described herein. In some embodiments, nucleic acids are circularized while being associated with, e.g. attached to, units. For example, a unit may be isolated within a microfluidic device, for example, in a channel or chamber of a microfluidic device, nucleic acids associated with the unit may be released from the unit may be circularized, e.g. through the use of an adaptor. In some embodiments, the circularized nucleic acids are attached to the unit, e.g. covalently or through affinity binding.

[0250] Adaptors, according to the various embodiments of the invention, can be extended using the circularized single stranded nucleic acid as a template. Alternatively, one or more different primers may be used to anneal elsewhere on the circle in addition or instead of the adaptor and can be extended with a polymerase enzyme. The extension reaction, such as rolling circle amplification, multi-primer rolling circle amplification or any other suitable extension reaction, can facilitate the creation of one long and linear single stranded amplicon nucleic acids comprising alternating replicas of the single stranded template nucleic acid and the adaptor hybridization sequences. In some embodiments, the combined replicas of the adaptor hybridization sequences are copies of the adaptor sequence, or differ by less than 8, 7, 6, 5, 4, 3, or 2 nucleotides. These sequences will together be referred to as “adaptor copies” for ease, but it is understood that they may refer to a number of different types of sequences generated from the extension reaction using the circle as a template. Nucleotide extension products produced using templates described herein, such as circularized single stranded nucleic acid templates, may be attached to units described herein. In some embodiments, nucleic acid extension products are attached to the same unit as the template nucleic acid that is used as a template to produce the nucleic acid extension product.

[0251] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved using the methods described herein or by any suitable method known in the art. For example, extension and / or amplification products may be cleaved at the 5′ end of a recognition site by Type II endonucleases. The cutting site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more upstream from the first nucleotide of the recognition site. The 5′ or 3′ end of a recognition site may form a 0-, 1-, 2-, 3-, 4-, or 5-nucleotide overhang. Blunt Type II endonucleases which cleave with a 0-nucleotide overhang include Mlyl and Schl. Exemplary Type IIS endonucleases which generate 5′ overhangs (e.g., 1, 2, 3, 4, 5 nucleotides overhangs) include, but are not limited to, Alwl, Bed, BceAI, BsmAI, BsmFI, Fokl, Hgal, Plel, SfaNI, BfuAI, Bsal, BspMI, BtgZI, Earl, BspQI, Sapl, Sgel, BceFI, BslFI, BsoMAI, Bst71I, Faql, Acelll, BbvII, Bvel, and Lgul. Nicking endonucleases which remove the recognition site and cleave on the 5′ site of the recognition site include, but are not limited to Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, Finl, Tsui, UbaFl 1I, Unbl, Vpakl 1AI, BspGI, Drdll, Pfll 108I, and UbaPI.

[0252] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved by non-Type IIS endonucleases which cleave at the 5′ end of the recognition site on both strands to generate a blunt end. The amplification product may be cleaved by non-Type IIS endonucleases which cleave at the 5′ end of the recognition site on one strand and in the middle of the recognition site on the other strand, generating a 5′ overhang. Examples of endonucleases which generate a 5′ overhang include, but are not limited to, BfuCI, DpnII, Fatl, Mbol, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I, Tsp45I, Aoxl, BscFI, Bspl43I, BssMI, BseENII, BstMBI, Kzo9I, Nedll, Sse9I, Tasl, TspEI, Ajnl, BstSCI, EcoRII, Maelll, NmuCI, and Psp6I.

[0253] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved by nicking endonucleases which cleave at the 5′ end of a recognition site to produce a nick. The nicking site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more upstream from the first nucleotide of the recognition site. Exemplary nicking endonucleases include, but are not limited to, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, Finl, Tsui, UbaF 1I, Unbl, Vpakl 1AI, BspGI, Drdll, Pfll 108I, and UbaPI.

[0254] Nucleic acids, e.g. the extension and / or amplification products produced from template nucleic acids described herein, may be cleaved at the 3′ end of a recognition site by Type IIS endonucleases. The 5′ or 3′ end of a recognition site may form a 0-, 1-, 2-, 3-, 4-, or 5-nucleotide overhang. The cutting site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more downstream from the last nucleotide of the recognition site. Type IIS endonucleases which cleave at 0 nucleotides downstream of the last nucleotide of the recognition site include Mlyl and Schl. Exemplary Type IIS endonucleases which generate 3′ overhangs (e.g., 1, 2, 3, 4, 5 nucleotide overhangs) include, but are not limited to, Mnll, BspCNI, Bsrl, BtsCI, Hphl, HpyAV, MboII, Acul, BciVI, Bmrl, Bpml, BpuEI, BseRI, Bsgl, Bsml, BsrDI, Btsl, Ecil, Mmel, NmeAIII, Hin4II, TscAI, Bce83I, Bmul, Bsbl, and BscCI. Non-Type II endonucleases which remove the recognition site on one strand and generate a 3′ overhang or blunt end on the other strand include, but are not limited to Nlalll, Hpy99I, TspRI, Fael, Hinlll, Hsp92II, Setl, Tail, Tscl, TscAI, and TseFI. Nicking endonucleases which remove the recognition site and cut on the 3′ end of the recognition site include Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI.

[0255] The adaptor sequences described herein may comprise one or more restriction recognition sites. In some embodiments, the recognition site is at least 4, 5, or 6 base pairs long. In some embodiments, the recognition site is non-palindromic. In some embodiments, the adaptor oligonucleotide comprises two or more recognition sites. Two or more recognition sites may be cleaved with one or more restriction enzymes. Exemplary pairs of recognition sites in an adaptor sequence include, but are not limited to, Mlyl-Mlyl, Mlyl-Nt.AlwI, Bsal-Mlyl, Mlyl-BciVI, and BfuCI-Mlyl.Sequencing

[0256] In any of the embodiments, the detection or quantification analysis of polynucleotides described herein, e.g. polynucleotides synthesized on units described herein or polynucleotides captured thereby or produced based thereon, such as amplification products using such polynucleotides as templates, can be accomplished by sequencing. The subunits or entire synthesized oligonucleotides can be detected via full sequencing of all oligonucleotides by any suitable methods known in the art, e.g., Illumina HiSeq 2500, including the sequencing methods described herein.

[0257] Sequencing can be accomplished through classic Sanger sequencing methods which are well known in the art. Sequencing can also be accomplished using high-throughput systems some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, i.e., detection of sequence in real time or substantially real time.

[0258] In some embodiments, high-throughput sequencing involves the use of next-generation sequencing (NGS) technology available by Illumina Genome Analyzer II, MiSeq personal sequencer, or HiSeq systems, such as those using HiSeq 4000, HiSeq 3000, HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000.

[0259] In some embodiments, NGS comprises the use of technology available by ABI SOLiD System. This genetic analysis platform may be used for massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology may utilize sequential ligation with dye-labeled oligonucleotides.

[0260] The NGS may comprise ion semiconductor sequencing (e.g., using technology from formerly Life Technologies, now Thermo Fisher (Ion Torrent)). Ion semiconductor sequencing can take advantage of the fact that when a nucleotide is incorporated into a strand of DNA, an ion can be released.

[0261] In some embodiments, NGS comprises the use of the Single Molecule Sequencing by Synthesis (SMSS) method, discussed in further detail in U.S. Pub. Nos. 2006 / 002471 I; 2006 / 0024678; 2006 / 0012793; 2006 / 0012784; and 2005 / 0100932.

[0262] High-throughput sequencing of oligonucleotides can be achieved using any suitable sequencing method known in the art, such as those commercialized by Pacific Biosciences, Complete Genomics, Genia Technologies, Halcyon Molecular, Oxford Nanopore Technologies and the like. The sequencing of polynucleotides may be performed using a next generation sequencing technique, e.g. real-time (SMRT™) technology by Pacific Biosciences. In some embodiments, the next generation sequencing comprises nanopore sequencing (See e.g., Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001). The nanopore sequencing technology from Oxford Nanopore Technologies; e.g., a GridlON system is used in various embodiments. Nanopore sequencing technology from Roche (formerly GENIA) can be used. The next generation sequencing may comprise DNA nanoball sequencing (as performed, e.g., by Complete Genomics; see e.g., Drmanac et al. (2010) Science 327: 78-81).Genes

[0263] The methods and compositions of the invention in various embodiments allow for the construction of gene libraries comprising a collection of individually accessible polynucleotides of interest. The polynucleotides can be linear, can be maintained in vectors (e. g., plasmid or phage), cells (e. g., bacterial cells), as purified DNA, or in other suitable forms known in the art. Library members (variously referred to as clones, constructs, polynucleotides, etc.) can be stored in a variety of ways for retrieval and use, including for example, in multiwell culture or microtiter plates, in vials, in a suitable cellular environment (e.g., E. coli cells), as purified DNA compositions on suitable storage media (e.g., the Storage IsoCodeD ID™ DNA library card; Schleicher & Schuell Bioscience), or a variety of other suitable library forms known in the art. A gene library may comprise about or at least 10, 100, 200, 300, 400, 500, 600, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7500, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 75000, 100000 members, or more.

[0264] In various embodiments, the methods and compositions of the invention allow for production of synthetic (i.e. de novo synthesized) genes. Libraries comprising synthetic genes may be constructed by a variety of methods described in further detail elsewhere herein, such as PCA, non-PCA gene assembly methods or hierarchical gene assembly, combining (“stitching”) two or more double-stranded polynucleotides (referred to here as “synthons”) to produce larger DNA units (i.e., multisynthons or chassis). Libraries of large constructs may comprise polynucleotides that are about or at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb long or longer. The large constructs may be shorter than 50000, 20000, 10000 or 5000 base pairs. The synthesis of any number of polypeptide-segment encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, as well as non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest is contemplated according to the embodiments of the invention. The term “gene” as used herein may refer broadly to any type of coding or non-coding, long polynucleotide or polynucleotide analog.

[0265] In some embodiments, nucleic acids and / or nucleic acid libraries described herein comprise genes encoding for a part or all of the genome of a synthetic organism, e.g. a virus or a bacterium. The terms “gene”, “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a nucleotide polymer. Unless otherwise limited, the same include known analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides. They can be of polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more of the modified nucleotides described herein or any other modified nucleotides known in the art, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0266] The term nucleic acid encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0267] The term nucleic acid also encompasses any chemical modification thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrosta...

Examples

example 1

Positional Encoding Device Architecture

[0445]We constructed a system configured to perform loading, holding, and manipulating of units as an example of positional encoding within a microfluidic device. The system comprises a fluidic network and a flow control system that controls the fluid flow through the network, as depicted in FIG. 14. The fluidic network is constructed from fused silica capillaries (363 um OD, 50 um ID, Molex), capillary tubing connectors (CapTight connectors, LabSmith), and custom fabricated connectors.

[0446]The bead-containing portion of the network begins with a feeder channel 1405 that serves as both a loading channel and a repository for beads prior to bead rearrangement. This channel was connected to a main channel 1410 through a custom-fabricated T-connector 1406 that serves as a bead spacer. Two branch channels 1412, 1420 were connected to the main channel via additional T-connectors that were configured to service as bead spacers. Beads may be distribut...

example 2

Positional Encoding Device—Bead Spacer

[0450]We first manually loaded a set of highly monodisperse 40 μm beads into the feeder channel 1405, capped the channel input with a bead stop 1404, and connected the other side of the bead stop to the channel's fluid control line 1403. Then, we directed flow in the main channel toward the top side of the main channel 1410, 1418, 1426 and applied pressure to the feeder channel via the reservoir 1416 and the main channel reservoir 1417.

[0451]Beads were fed through the feeder channel in a stacked regime. When abutting beads reached the T-connector, the cross-flow created separation between the beads as they entered the main channel 1410.

[0452]Snapshot images from a movie of beads being separated using a T-connector are shown in FIG. 23. We developed a bead spacer to address the challenges of manipulating beads within the stacked regime (i.e., risks of clogging and loss of positional encoding at changes in channel dimension, and difficulty sorting...

example 3

Positional Encoding Device—Bead Distributing

[0458]Beads within the main channel 1410 are flowed towards the branch channels 1412, 1420. We distribute beads into branch channels by adjusting the applied pressure on the main channel upstream and downstream of each branch point 1411, 1419 and by selectively activated flow within the branch channels 1412, 1420 via the two-way selector valve 1433 such that the carrier fluid distributed each bead into its preassigned branch channel. After a first bead enters its designated branch channel, the subsequent pressure configuration and branch channel activation is determined by the branch assignment of the next bead to be distributed. If this second bead is designated for the same branch channel, the applied pressures and the two-way selector valve setting is kept the same. On the other hand, if the second bead was designated for the other branch channel, we adjust the pressures on the main channel and the flow activation of branch channels in ...

Claims

1. A method of synthesizing oligomers associated with mobile units, the method comprising:(a) routing k mobile units through a first channel of a microfluidic device in a first order;(b) distributing at least a subset of the k mobile units into at least z branch channels; and(c) routing the at least a subset of the k mobile units from the at least z branch channels into the first channel in a second order;wherein at least a subset of the k mobile units are functionalized with a group suitable to synthesize an oligomer; wherein at least a subset of the k mobile units are mappable to a path comprising a specific one of the z branch channels; wherein at least a subset of the k mobile units are subjected to reaction conditions comprising conditions for a step of a synthesis reaction inside the z branch channels;and wherein steps a-c are repeated for n cycles, wherein n is at least 2 and z is at least 2.

2. The method of claim 1, wherein the synthesis reaction comprises a nucleic acid synthesis reaction or a peptide synthesis reaction.

3. The method of claim 2, wherein the nucleic acid synthesis reaction is a template independent nucleic acid synthesis reaction.

4. The method of claim 1, wherein the reaction conditions comprise an enzyme.

5. The method of claim 4, wherein the enzyme is selected from a terminal deoxynucleotidyl transferase, a thermostable DNA polymerase, a DNA polymerase theta, a Poly (A) polymerase, and a DNA polymerase encoded by a variant of the 9°N DNA Polymerase gene from Thermococcus species 9°N-7.

6. The method of claim 5, wherein the variant of the 9°N DNA Polymerase gene comprises the 9°N (D141A / E143A / A485L) DNA Polymerase gene or the 9°N (E143D) DNA Polymerase gene.

7. The method of claim 4, wherein the enzyme is conjugated to a nucleotide or a nucleotide analog.

8. The method of claim 1, wherein the reaction conditions comprise a nucleotide or a nucleotide analog.

9. The method of claim 1, wherein at least a subset of the k mobile units are functionalized with an initiator nucleic acid or a nascent oligonucleotide.

10. The method of claim 1, wherein the synthesis reaction inside the z branch channels comprises performing a coupling reaction by catalyzing formation of a covalent bond between a terminal nucleotide of initiator nucleic acids or nascent oligonucleotides associated with at least a subset of the k mobile units and a new nucleotide or nucleotide analog in the presence of a transferase enzyme.

11. The method of claim 10, wherein the new nucleotide or nucleotide analog comprises a blocking moiety.

12. The method of claim 11, further comprising performing a deblocking reaction thereby removing the blocking moiety from the newly incorporated nucleotide or nucleotide analog.

13. The method of claim 10, further comprising one or more steps selected from the group consisting of a washing step, a modification step, a cleaving step, and a capping step.

14. The method of claim 13, wherein two or more of the steps selected from the group consisting of the coupling reaction, the deblocking reaction, the washing step, the modification step, the cleaving step, and the capping step are performed in different cycles.

15. The method of claim 1, wherein the oligomers are oligonucleotides and wherein the method further comprises assembling the oligonucleotides into genes.

16. The method of claim 1, wherein the reaction conditions comprise one or more of reagents selected from the group consisting of an amino acid, a dipeptide, a polypeptide, and a carbodiimide.

17. The method of claim 16, further comprising performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal end of nascent peptides associated with at least a subset of the k mobile units and a new amino acid, dipeptide or polypeptide.

18. The method of claim 17, further comprising performing one or more step selected from the group consisting of a capping step, a washing step, and a deprotecting step.

19. The method of claim 18, wherein two or more of the steps selected from the group consisting of the coupling reaction, capping step, washing step and the deprotecting step are performed in different cycles.

20. The method of claim 1, wherein the same z branch channels are used in at least two of the n cycles.

21. The method of claim 1, wherein k is at least 2.

22. The method of claim 1, wherein(a) the mobile units are selected from the group consisting of beads, droplets, cells, bubbles, slugs, immiscible volumes, glass beads, polymer beads, cross-linked beads, cross-linked polymer beads, divinylbenzene cross-linked polymer beads, and divinylbenzene cross-linked polystyrene beads; and / or(b) the first order is different in at least two of the n cycles; and / or(c) the second order is different in at least two of the n cycles.

Images